Method of producing a taxane-type diterpene and a method of obtaining cultured cells which produce the taxane-type diterpene at a high rate

ABSTRACT

This invention relates to a method of producing a taxane-type diterpene(s) wherein tissues or cells of a plant which produces taxane-type diterpene(s) is cultured in the presence of at least one selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents, a method of producing a taxane-type diterpene wherein the tissues or the cells of the plant are cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture, a method of producing a taxane-type diterpene wherein the tissue or the cell of the plant is cultured in a culture vessel, while oxygenic gas containing 0.03-10% of carbon dioxide is used as aeration gas to be introduced to the vessel, and a method of obtaining highly productive cultured cells for the taxane-type diterpene wherein cultured cells of the plant which produces the taxane-type diterpene are separated into a plurality of layers according to the difference in their specific gravities, and the cells contained in at least one layer are cultured, then highly productive cultured cells for the taxane-type diterpene are selected from among those cultured cells. 
     The present invention allows industrial production of a taxane-type diterpene such as taxol which is useful as a therapeutic agent for cancer.

This application is a divisional of application Ser. No. 08/491,844,filed on Jul. 14, 1995 now U.S. Pat. No. 5,637,484, the entire contentsof which are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to a method of producing a taxane-type diterpeneincluding taxol which is useful as a therapeutic agent for ovariancancer, mammary cancer, lung cancer and the like, and a method ofobtaining cultured cells which produce the taxane-type diterpene at ahigh rate.

BACKGROUND ART

Taxol, which is useful as a therapeutic agent for ovarian cancer,mammary cancer, lung cancer and the like, is a taxane-type diterpeneidentified after being isolated from Taxus brevifolia NUTT, which is aplant belonging to genus Taxus, family Taxaceae and has a complex estergroup which is related to its activity. Taxol can be found in all theparts of the plant body of Taxus brevifolia NUTT, but the bark has beenreported to exceed all others in its content of the taxol. At present,taxol is collected from a natural or a cultivated plant body, however,the plant belonging to genus Taxus grows slowly, and it takes more than10 years to grow to a height of 20 cm above the ground, besides the treedies after its bark is taken off, thus it has been difficult to easilyobtain a large amount of taxol. It would be advantageous if ataxane-type diterpene such as taxol and baccatin III which is aprecursor of taxol, can be produced by the use of tissue culture, sincea large amount of taxol can be easily obtained without cutting down thetrees.

As a conventional method of producing taxol by utilizing cultured plantcells, a US patent was issued on a production method utilizing culturedcells of Taxus brevifolia NUTT (U.S. Pat. No. 5,019,504), however, theyield of taxol production described therein is 1-3 mg/l, and that isinsufficient for the industrial production. Besides, the production oftaxol by the cell culture is unstable and even when a primary cell ofhigh productivity can be obtained by selection, it is difficult to keepits content by subculturing [E. R. M. Wickremesine et al., WorldCongress on Cell and Tissue Culture (1992)].

On the other hand, as a prior art in the taxol production, asemisynthetic method from baccatin III, which is a precursor inbiosynthesis of taxol, is disclosed in the specification of U.S. Pat.No. 5,015,744 issued to Holton et al. By the use of the plant tissueculture, a raw material for the semisynthetic process such as baccatinIII can be produced, thus the plant tissue culture can be also utilizedfor taxol production by the above-mentioned semisynthetic process.

DISCLOSURE OF INVENTION

The first object of the present invention is to provide a simple methodof producing a taxane-type diterpene by plant tissue culture.

The second object of the present invention is to provide a method ofobtaining cultured cells which produce a taxane-type diterpene at a highrate.

The first invention of the present application is a method of producinga taxane-type diterpene wherein a tissue or a cell of a plant whichproduces a taxane-type diterpene is cultured in the presence of at leastone substance selected from the group consisting of jasmonic acids,compounds containing a heavy metal, complex ions containing a heavymetal, heavy metal ions, amines and antiethylene agents, then thetaxane-type diterpene is recovered from the resulting cultures.

The second invention of the present application is a method of producinga taxane-type diterpene wherein a tissue or a cell of a plant whichproduces a taxane-type diterpene is cultured by controlling the oxygenconcentration in a gas phase in a culture vessel to less than the oxygenconcentration in the atmosphere, from the initial stage of the culture,or by controlling the dissolved oxygen concentration in a fluid mediumwhich is in contact with the tissue or the cell to less than thesaturated dissolved oxygen concentration at that temperature, from theinitial stage of the culture, then the taxane-type diterpene isrecovered from the resulting cultures.

The third invention of the present application is a method of obtainingcultured cells which produce a taxane-type diterpene at a high rate,wherein cells of a plant which produces a taxane-type diterpene arefractionated into a plurality of layers according to the difference intheir specific gravities, and cells contained in at least one layer arecultured, then such cultured cells that produce the taxane-typediterpene at a high rate are selected from among those cultured cells.

The present invention will be described in further detail.

The taxane-type diterpene, which is an object for the present invention,is not particularly limited to any diterpene as far as it has a taxaneskeleton, and the illustrative examples include taxol, 7-epitaxol,baccatin III, 7-epibaccatin III, cephalomannine, 7-epicephalomannine,10-deacetylbaccatin III, 10-deacetylcephalomannine, 10-deacetyltaxol,taxagifine, an analogue thereof, taxane 1a, an analogue thereof, xylosylcephalomannine, xylosyl taxol and the like.

Examples of the plant to be used in the present invention which producesthe taxane-type diterpene are those belonging to genus Taxus, such asTaxus baccata LINN, Taxus cuspidata SIEB. et ZUCC, Taxus cuspidata SIEB.et ZUCC var. nana REHDER, Taxus brevifolia NUTT, Taxus canadiensisMARSH, Taxus chinensis, and Taxus media.

According to the first invention of the present application, culture ofthe above-mentioned plant can be carried out by the previously knownmethod except that the tissue or the cell of the plant which producesthe taxane-type diterpene is cultured in the presence of at least onesubstance selected from the group consisting of jasmonic acids,compounds containing a heavy metal, complex ions containing a heavymetal, heavy metal ions, amines and antiethylene agents.

Examples of jasmonic acids, which are objects for the first invention ofthe present application, include a compound represented by the generalformula (I): ##STR1## [wherein, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e)and R^(1f) respectively represent hydrogen atom, hydroxyl group, alkylgroup having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbonatoms;

R², R³, R⁴, R⁵ and R^(6a) respectively represent hydrogen atom or alkylgroup having 1 to 6 carbon atoms;

a side chain consisting of C¹ --C² --C³ --C⁴ --C⁵ --C⁶ may contain oneor more double bonds;

R^(6b) represents hydroxyl group or --O-- carbohydrate residue;

R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸ represents hydrogenatom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6carbon atoms or amino acid residue), OR⁹ (wherein R⁹ is alkyl grouphaving 1 to 6 carbon atoms or carbohydrate residue), or alkyl grouphaving 1 to 6 carbon atoms;

n is an integer of 1-7;

and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms],

a compound represented by the general formula (II): ##STR2## [wherein,R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) and R^(1f) respectively representhydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms,or alkoxy group having 1 to 6 carbon atoms;

R², R³, R⁴, R⁵ and R⁶ respectively represent hydrogen atom or alkylgroup having 1 to 6 carbon atoms;

a side chain consisting of C¹ --C² --C³ --C⁴ --C⁵ --C⁶ may contain oneor more double bonds;

R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸ represents hydrogenatom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6carbon atoms or amino acid residue), OR⁹ (wherein R⁹ is alkyl grouphaving 1 to 6 carbon atoms or carbohydrate residue), or alkyl grouphaving 1 to 6 carbon atoms;

n is an integer of 1-7;

and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms],

and a compound represented by the general formula (III): ##STR3##[wherein, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) and R^(1f) respectivelyrepresent hydrogen atom, hydroxyl group, alkyl group having 1 to 6carbon atoms, or alkoxy group having 1 to 6 carbon atoms;

R², R³, R⁴, R⁵ and R⁶ respectively represent hydrogen atom or alkylgroup having 1 to 6 carbon atoms;

a side chain consisting of C¹ --C² C³ --C⁴ --C⁵ --C⁶ may contain one ormore double bonds;

R⁷ represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR⁸ (wherein R⁸ represents hydrogenatom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6carbon atoms or amino acid residue), OR⁹ (wherein R⁹ is alkyl grouphaving 1 to 6 carbon atoms or carbohydrate residue), or alkyl grouphaving 1 to 6 carbon atoms;

n is an integer of 1-7;

and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms].

Preferable examples of jasmonic acids represented by the above-mentionedgeneral formula (I) include a compound represented by the generalformula (I'): ##STR4## [wherein, R^(1') represents hydrogen atom orhydroxyl group; a side chain consisting of C¹ --C² --C³ --C⁴ --C⁵ --C⁶may contain a double bond between C¹ and C², between C² and C³, orbetween C³ and C⁴ ; R^(6b) represents hydroxyl group or --O--carbohydrate residue;

R^(7') represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR^(8') (wherein R^(8') representshydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having1 to 4 carbon atoms or amino acid residue) or OR^(9') (wherein R^(9')represents alkyl group having 1 to 4 carbon atoms or carbohydrateresidue);

n is an integer of 1-7;

and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms], and preferableexamples of jasmonic acids represented by the above-mentioned generalformula (II) include a compound represented by the general formula(II'): ##STR5## [wherein, R^(1') represents hydrogen atom or hydroxylgroup; a side chain consisting of C¹ --C² --C³ --C⁴ --C⁵ --C⁶ maycontain a double bond between C¹ and C², between C² and C³, or betweenC³ and C⁴ ;

R^(7') represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR^(8') (wherein R^(8') representshydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having1 to 4 carbon atoms or amino acid residue) or OR^(9') (wherein R^(9')represents alkyl group having 1 to 4 carbon atoms or carbohydrateresidue);

n is an integer of 1-7;

and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms], and preferableexamples of jasmonic acids represented by the above-mentioned generalformula (III) include a compound represented by the general formula(III'), ##STR6## [wherein, R^(1') represents hydrogen atom or hydroxylgroup; a side chain consisting of C¹ --C² --C³ --C⁴ --C⁵ --C⁶ maycontain a double bond between C¹ and C², between C² and C³, or betweenC³ and C⁴ ;

R^(7') represents hydroxyl group, OM (wherein M is alkali metal atom,alkaline earth metal atom or NH₄), NHR^(8') (wherein R^(8') representshydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having1 to 4 carbon atoms or amino acid residue) or OR^(9') (wherein R^(9')represents alkyl group having 1 to 4 carbon atoms or carbdhydrateresidue);

n is an integer of 1-7;

and in the above-mentioned five-membered ring, a double bond may beformed between the neighboring member carbon atoms].

In the above-mentioned general formulae (I), (II) and (III), examples ofalkyl group having 1 to 6 carbon atoms represented by R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵, R⁶, R^(6a), R⁷, R⁸ or R⁹include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl groups.

In the above-mentioned general formulae (I), (II) and (III), examples ofalkoxy group having 1 to 6 carbon atoms represented by R^(1a), R^(1b),R^(1c), R^(1d), R^(1e) or R^(1f) include, for example, methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy,n-pentyloxy and n-hexyloxy groups.

When R⁷ is OM, examples of an alkali metal atom or an alkaline earthmetal atom represented by M include, for example, sodium, potassium andcalcium.

When R⁷ is NHR⁸, the acyl group having 1 to 6 carbon atoms representedby R⁸ may have either a straight chain or a branched chain, and theirexamples include, for example, formyl, acetyl, propionyl, butyryl,valeryl, hexanoyl and acryloyl groups.

When R⁷ is NHR⁸, examples of an amino acid residue represented by R⁸include isoleucyl, tyrosyl, and tryptophyl groups.

When R⁷ is OR⁹, an example of a carbohydrate residue represented by R⁹is glucopyranosyl group, and when R^(6b) is --O-- carbohydrate residuein the above-mentioned general formula (I), an example of a carbohydrateresidue is glucopyranosyl group.

In the compounds represented by the general formulae (I), (II) and(III), a double bond may be formed between the neighboring member carbonatoms in the five-membered ring.

Illustrative examples of the compound represented by the general formula(I) include those shown as follows; ##STR7##

Illustrative examples of the compound represented by the general formula(II) include those shown as follows; ##STR8##

Illustrative examples of the compound represented by the general formula(III) include those shown as follows;

(Compound I)

R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵, R⁶ : H

A double bond is formed between C³ and C⁴.

R^(7a) : --OH or --OCH₃

n: 1 to 3

(Compound J)

R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R², R³, R⁴, R⁵, R⁶ :H

R⁷ : --OH

n: 1

Illustrative examples of the compound represented by the general formula(III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e) or R^(1f) ishydroxyl group, or a double bond is formed between the neighboringmember carbon atoms in the five-membered ring, include those shown asbelow; ##STR9##

Preferable examples of a compound represented by the general formula(I), (II) or (III) include the compounds wherein R^(1a), R^(1b), R^(1c),R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵ and R⁶ are hydrogen atoms, R⁷ ishydroxyl group or methoxy group, and a side chain consisting of C¹ --C²--C³ --C⁴ --C⁵ --C⁶ does not contain a double bond, or contain a doublebond between C¹ and C², between C² and C³, or between C³ and C⁴.

Jasmonic acids to be used in the present invention which are representedby the general formula (I), (II) or (III) have various stereoisomers(cis-trans isomers and optical isomers), and each isomer can be usedalone or in the form of a mixture.

All of the jasmonic acids shown above have the effect of improving theproductivity in the taxane-type diterpene production, however, tuberonicacid, methyl tuberonate, cucurbic acid, methyl cucurbate, jasmonic acidand methyl jasmonate, which are the compounds represented by the generalformula (I), (II) or (III) wherein R^(1a), R^(1b), R^(1c), R^(1d),R^(1e), R^(1f), R², R³, R⁴, R⁵ and R⁶ are hydrogen atoms, R⁷ is hydroxylgroup or methoxy group, n is 1, and a double bond is formed between C³and C⁴, are particularly preferable from the view point of their higheffectiveness in improving the productivity.

These jasmonic acids are prepared by synthesis or extraction and thelike from a plant (H. Yamane et al: Agric. Biol. Chem., 44,2857-2864(1980)).

By the way, there is a description teaching that jasmonic acids areproduced by various plants by themselves as a phytohormone-likesubstance which induces various reactions related to growth promotion,maturation of tissue and appearance of resistance to disease (TeruhikoYoshihara. Shokubutsu Saibo Kogaku, Vol 2, No. 4 523-531 (1990)).

Accordingly, the jasmonic acids involved in the present invention, canbe not only added from outside of the culture system, but also producedby the cultured cells or cultured tissues by themselves. An example of amethod to promote the production of such endogenous jasmonic acids bythe cultured cells or cultured tissues includes addition ofmicroorganism cultures, an extract or a heat-treated substance thereof,or a plant extract to a culture medium, and an illustrative example ofsuch a method is a process of adding a fungus cell wall fractiondescribed by M. J. Mueller et al., Proc. Natl. Acad. Sci. U.S.A., 90(16), 7490-7494 (1993)). It is also possible to increase the amount ofthe produced endogenous jasmonic acid by partially damaging culturedcells or cultured tissues mechanically, or with ultraviolet rays, Orheat, and one illustrative example of such a process is mechanicalcytoclasis of a part of cells (R. A. Cleeman et al., Proc. Natl. Acad.Sci. U.S.A., 89(11), 4938-4941 (1989).

Since jasmonic acids are hardly soluble in water, they are usuallydissolved in an organic solvent such as ethanol and methanol ordissolved in a surfactant and the like, then added to a culture medium.Jasmonic acids in liberated form can be used as they are, or they areused in the form of a salt by being neutralized with an alkali.

Of the jasmonic acids, those compounds represented by the formula (I) or(III) tend to be in stable trans-form rather than unstable cis-form,since epimerization occurs at the alpha-position to the carbonyl groupin the five-membered ring by an acid, an alkali or heat. In anequilibrium experiment utilizing natural or synthesized jasmonic acids,the trans-form is present in the ratio of 90% and the cis-form ispresent in the ratio of 10%. Generally, cis-form is considered to have ahigher activity, but jasmonic acids to be used in the present inventioninclude all the stereoisomers of the compounds represented by theabove-mentioned formula (I) or (III), and the mixture thereof.

Jasmonic acids are required to have a concentration in a culture mediumof 0.01-1000 μM, and it is particularly preferable, according to thefirst invention of the present application, to control the concentrationof the jasmonic acids to be in the range of 0.1 to 500 μM.

Induction of some secondary metabolites by addition of jasmonic acids toplant cell cultures is described in DE 4122208 C1, however, there havebeen no reports on carrying out tissue culture of a taxane-typediterpene producing plant in the presence of jasmonic acids as a mediumadditive, and it has been beyond all expectations that the amount of theproduced taxane-type diterpene, which has totally different biosyntheticpathway or biosynthesis controlling mechanism from those of thesecondary metabolites disclosed in the above-mentioned patent, wasincreased by, the method of the first invention of the presentapplication.

There is a description in the International Publication WO No. 93/17121that jasmone or methyl jasmone, which has a structure analogous to thoseof jasmonic acids represented by the formula (I), (II) or (III) to beused in the present invention, is effective in induction of taxolproduction. However, these compounds do not have a group such ascarboxyl, which is represented by the formula: --(CH₂)_(n) --CO--R⁷ inthe formula (I), (II) or (III), unlike the jasmonic acids, and the taxolinducing activity of these compounds was found to be low (seeComparative Example No. 24).

The heavy metals, which are objects for the first invention of thepresent application, are not particularly limited to any heavy metal asfar as it belongs to the copper group or the iron group, however, as themetal belonging to the copper group, the use of silver is particularlypreferable, and as the metal belonging to the iron group, the use ofcobalt is particularly preferable. In addition to that, when silver orcobalt is used, it is preferably used in the form of a compoundcontaining the said heavy metal, a complex ion containing the said metalor in the form of the said metal ion. These compounds can be used aloneor in combination.

Illustrative examples of the compound containing silver include silvernitrate, silver sulfate, silver fluoride, silver chlorate, silverperchlorate, silver acetate, silver sulfite, silverhexafluorophosphate(V), silver tetrafluoroborate, diamine silver(I)sulfate, potassium diaminoargentate(I) and the like. Among these,particularly preferable compounds can be exemplified by silver nitrate,silver sulfate and the like.

Illustrative examples of the complex ion containing silver include[Ag(S₂ O₃)₂ ]³⁻, [Ag(S₂ O₃)₃ ]⁵⁻, [Ag(NH₃)₂ ]⁺, [Ag(CN)₂ ]⁻, [Ag(CN)₃]²⁻, [Ag(SCN)₂ ]⁻, [Ag(SCN)₄ ]³⁻ and the like. Among these, particularlypreferable complex ions can be exemplified by [Ag(S₂ O₃)₂ ]³⁻, [Ag(S₂O₃)₃ ]⁵⁻ and the like.

Illustrative examples of the compound containing cobalt include cobaltchloride, cobalt nitrate, cobalt sulfate, cobalt fluoride, cobaltperchlorate, cobalt bromide, cobalt iodide, cobalt selenate, cobaltthiocyanate, cobalt acetate, ammonium cobalt sulfate, cobalt(II)potassium sulfate, hexaamminecobalt(III) chloride,pentaammineaquacobalt(III)chloride,nitropentaamminecobalt(III) chloride,dichlorotetraamminecobalt(III) chloride hemihydrate,dinitrotetraamminecobalt(III) chloride, carbonatotetraamminecobalt(III)chloride, ammonium tetranitrodiamminecobaltate(III), sodiumhexanitrocobaltate(III), tris(ethylenediamine)cobalt(III) chloridetrihydrate, dichlorobis(ethylenediamine)cobalt(III) chloride, potassiumtris(oxalato)cobaltate(III) trihydrate, potassiumhexacyanocobaltate(III), potassium(ethylenediaminetetraacetato)cobaltate(III) dihydrate,hydridotetracarbonylcobalt(I), dicarbonyl(cyclopentadienyl)cobalt(I),octacarbonyldicobalt(O), hexacarbonyl(acetylene)dicobalt(O),bis(cyclopentadienyl)cobalt(I),(cyclopentadienyl)(1,5-cyclooctadiene)cobalt(I) and the like. Amongthese, particularly preferable compounds can be exemplified by cobaltchloride, cobalt nitrate, cobalt sulfate and the like.

Illustrative examples of the complex ion containing cobalt includepentaammineaquacobalt ion, nitropentaamminecobalt ion,dichlorotetraamminecobalt ion, dinitrotetraamminecobalt ion,carbonatotetraamminecobalt ion, tetranitrodiamminecobalt ion,hexanitrocobalt ion, tris(ethylenediamine)cobalt ion,dichlorobis(ethylenediamine)cobalt ion, tris(oxalato)cobalt ion,hexacyanocobalt ion, (ethylenediaminetetraacetato)cobalt ion and thelike.

Of the said heavy metals, the compound containing silver, the complexion containing silver or the silver ion preferably has a concentrationin the medium of 10⁻⁸ M-10⁻¹ M, and it is further preferable to adjustthe concentration to be in the range of 10⁻⁷ M to 10⁻² M. The compoundcontaining cobalt, the complex ion containing cobalt or the cobalt ionpreferably has a concentration in the medium of 10⁻⁶ -10⁻¹ M, and it isfurther preferable to adjust the concentration to be in the range of10⁻⁵ to 10⁻² M.

So far, there are no cases reported wherein the tissue culture of aplant producing a taxane-type diterpene is carried out in the presenceof a compound containing silver, a complex ion containing silver orsilver ion as an additive to the medium. Although compounds containingcobalt, or cobalt ions are contained as one of the medium components forsuch a medium that is generally used as a medium for the tissue cultureof a plant belonging to genus Taxus, such as Linsmaier-Skoog medium,Murashige-Skoog medium, and Gamborg's B-5 medium, they are used at aconcentration of 1×10⁻⁷ M-4×10⁻⁷ M [Growth and breeding of a woodyplant, edited by the latest biotechnology complete works editorscommittee, Nogyo Tosho, P265-268], which is a much lower concentrationthan those used in the method of the present invention. In the meantime,there are no cases reported wherein the tissue culture of a plantproducing a taxane-type diterpene is carried out in the presence of acompound containing cobalt or cobalt ions of such a high concentrationthat is used in the first invention of the present application, justlike the case with the above-mentioned silver compound. In addition tothat, it was beyond all expectations that the amount of the taxane-typediterpene to be produced is increased by the culture carried out in thepresence of such heavy metals.

According to the first invention of the present application, by amineswe refer to an amine or a salt thereof. As the amines, which are theobjects for the first invention of the present application, bothmonoamines and polyamines can be used, however, the use of polyamines isparticularly preferable.

In addition to that, examples of the amines, which are the objects forthe first invention of the present application, include mono, di ortrialkyl amines wherein a part of hydrogen atoms in the alkyl group maybe substituted by hydroxyl group, such as methyl amine, ethyl amine,dimethyl amine, diethyl amine, triethyl amine, diethanol amine,triethanol amine or a salt thereof; polymethylene diamine wherein thepolymethylene moiety may be interrupted by imino group, and H in theamino group can be substituted by lower alkyl group, such as putrescine,cadaverine, spermidine, spermin, ethylenediamine,N,N-diethyl-1,3-propane diamine, triethylene tetramine, or a saltthereof; cyclic alkyl amine such as cyclopentyl amine, cyclohexyl amineor a salt thereof, or a cyclic amine such as methenamine and piperazine,or a salt thereof. Among these amines, preferable amines can beexemplified by polyamines such as putrescine [NH₂ (CH₂)₄ NH₂ ],cadaverine [NH₂ (CH₂)₅ NH₂ ], spermidine [NH₂ (CH₂)₃ NH(CH₂)₄ NH₂ ],spermin [NH₂ (CH₂)₃ NH(CH₂)₄ NH(CH₂)₃ NH₂ ], ethylene diamine [NH₂(CH₂)₂ NH₂ ], N,N-diethyl-1,3-propane diamine [(C₂ H₅)₂ N(CH₂)₃ NH₂ ],diethylene triamine [NH₂ (CH₂)₂ NH(CH₂)₂ NH₂ ] and the like or a saltthereof.

The said amines preferably have a concentration in the medium of 10⁻⁸M-10⁻¹ M, and it is further preferable to adjust the concentration to bein the range of 10⁻⁷ M to 10⁻² M.

One illustrative example wherein a secondary metabolite is shown to beinduced by addition of amines to the plant tissue cultures is shown inJapanese Patent Laid-Open Publication No. 4-262788 wherein indolealkaloid production is shown to be induced by addition of amines tocultured cells of Catharanthus roseus. However, there are no casesreported wherein the tissue culture of a plant producing taxane-typediterpene, which is a different plant species from that of Catharanthusroseus, was carried out in the presence of amines as an additive to themedium, and it was beyond all expectations that the amount of thetaxane-type diterpene, which has a totally different biosyntheticpathway from that of the indole alkaloid, to be produced can beincreased thereby.

The antiethylene agent, which is an object for the first invention ofthe present application is not particularly limited to any specificsubstance as far as it is a substance which inhibits the ethylenebiosynthesis mechanism of the cultures and/or a substance which removesthe ethylene remaining in the cultures or existing in the gas phase orin the medium in the culture vessel containing the cultures.

Illustrative examples of a method to inhibit the ethylene biosynthesismechanism include a method of inhibiting the activity of an enzyme whichcatalyzes the conversion of S-adenosyl-methionine into1-aminocyclopropane-1-carboxylic acid, and a method of inhibiting theactivity of an enzyme which catalyzes the conversion of1-aminocyclopropane-1-carboxylic acid into ethylene, and illustrativeexamples of the compound having the former function include,aminoxyacetic acid, acetylsalicylic acid, Rhizobitoxine,aminoethoxyvinylglycine, methoxyvinylglycine, a-aminoisobutyric acid,2,4-dinitrophenol and the like. They can also include a salt, an ester,an amino acid derivative and a carbohydrate derivative of the saidcompound.

Illustrative examples of the salt include sodium, potassium, calcium,and magnesium salts, illustrative examples of the ester include methyl,ethyl, propyl, and butyl esters, illustrative examples of the amino acidderivatives include glycine, methionine, and phenylalanine derivatives,and illustrative examples of the carbohydrate derivative include glucoseand maltose derivatives. The salt, ester, amino acid derivative,carbohydrate derivative according to the present invention are notlimited to the above-mentioned compounds.

Illustrative examples of the compound having the latter function includegallic acid, a salt, an ester, an amino acid derivative and acarbohydrate derivative thereof [Hiroshi Hyodo, Society of HorticultureAutumn Convention 1987 Symposium Summary, p. 122, Susumu Kuraishi,Phytohormone, Tokyo University Publication, p.111].

Illustrative examples of the salt include sodium, potassium, calcium,and magnesium salts, illustrative examples of the ester include methyl,ethyl, propyl, and butyl esters, illustrative examples of the amino acidderivatives include glycine, methionine, and phenylalanine derivatives,and illustrative examples of the carbohydrate derivatives includeglucose and maltose derivatives. The salt, ester, amino acid derivative,carbohydrate derivative according to the present invention are notlimited to the above-mentioned compounds.

Illustrative examples of the substance which removes the ethyleneremaining in the cultures or existing in the gas phase or the medium inthe culture vessel containing the cultures include 1,5-cyclooctadieneand isothiocyanic acid, a salt, an ester (such as allyl isothiocyanateand benzyl isothiocyanate), an amino acid derivative and a carbohydratederivative thereof [Megumi Munakata, Chemical control in plants, 29(1),89-93 (1994)].

Illustrative examples of the salt include sodium, potassium, calcium,and magnesium salts, illustrative examples of the ester include methyl,ethyl, propyl, butyl, and allyl esters, illustrative examples of theamino acid derivatives include glycine, methionine, and phenylalaninederivatives, and illustrative examples of the carbohydrate derivativesinclude glucose and maltose derivatives. The salt, ester, amino acidderivative, carbohydrate derivative according to the present inventionare not limited to the above-mentioned compounds.

The antiethylene agent is required to have a concentration in a culturemedium of 10⁻⁸ M-10⁻¹ M, and it is particularly preferable to controlthe concentration of the antiethylene agent to be in the range of 10⁻⁷ Mto 10⁻² M.

It is known that ethylene is one of phytohormones, and involved invarious physiological phenomena caused in the plant, such as growth ofindividium, morphogenesis, and aging. A report by Kim, Dong II et al.,Biotechnol. Bioeng., 38(4), 331-339 (1991) is an illustrative examplewherein ethylene is utilized for improving the productivity of thesecondary metabolite by the plant. However, in all the examples whereincontrolling of ethylene is utilized for improving the productivity ofthe secondary metabolite, it is the control of ethylene supply to theplant tissue cultures, as typically shown in the above-mentioned report,and so far there have been no cases reported in which the control toinhibit the ethylene production is utilized to improve the production ofthe secondary metabolite, like the method of the present invention.

In addition to that, the antiethylene agent is generally utilized as afreshness keeping agent for flowers, fruits and vegetables, however,there have been no cases reported wherein the antiethylene agent is usedfor the purpose of improving the production of the secondary metabolite.

Under these circumstances, the present inventors ascertained thatethylene greatly inhibits the production of the taxane-type diterpene bythe tissues and the cells of the taxane-type diterpene producing plant.Accordingly, based on the above-mentioned finding, the inventorscultured the said tissue cultures in the presence of the antiethyleneagent, and found out that the antiethylene agent not only controls theabove-mentioned inhibition but also remarkably improves the amount ofthe taxane-type diterpene resulting from the cultures. There have beenno cases reported wherein the production of the taxane-type diterpene isinduced by culturing the tissue cultures of a plant producingtaxane-type diterpene in the presence of an antiethylene agent, and itwas beyond all expectations that the productivity of the above-mentionedsecondary metabolite can be even increased by the method of the firstinvention of the present application.

Examples of the medium to be used for the first invention of the presentapplication include those known media which have been conventionallyused for the plant tissue culture, such as medium of Murashige & Skoog(1962), medium of Linsmaier Skoog (1965), Woody Plant Medium (1981),Gamborg's B-5 medium and Mitsui's M-9 medium.

A phytohormone, and if necessary a carbon source, an inorganiccomponent, vitamins, amino acids and the like may be added as well tothese media.

As a carbon source, a disaccharide such as sucrose, maltose, andlactose, a monosaccharide such as glucose, fructose and galactose,starch or a mixture of two or more kinds of such sugar sources mixed atan appropriate ratio can be utilized.

As an inorganic component, illustrative examples include phosphorus,nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc,boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, andthese components can be added in the form of such a compound aspotassium nitrate, sodium nitrate, calcium nitrate, potassium chloride,potassium monohydrogenphosphate, potassium dihydrogenphosphate, calciumchloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferricsulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate,sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chlorideand the like.

As the phytohormone, for example, auxin such as indoleacetic acid (IAA),naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid(2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can beused.

As the vitamins, for example, biotin, thiamin (vitamin B1), pyridoxine(vitaminB6), pantothenic acid, inositol, nicotinic acid and the like canbe used.

As the amino acids, for example, glycine, phenylalanine, leucine,glutamine, cysteine and the like can be added.

Generally, the carbon source in a concentration of about 1-about 30 g/l,the inorganic component in a concentration of about 0.1 μM-about 100 mM,the phytohormones in a concentration of about 0.01-about 10 μM, and thevitamins and the amino acids respectively in a concentration of about0.1-about 100 mg/l are used.

According to the present invention, both a liquid medium and such asolid medium that contains agar and gelan gum normally in an amount of0.1-1% can be used, however, usually a liquid medium is preferable.

According to the tissue culture of the present invention, a piece of atissue or a cell of a root, a growing point, a leaf, a stem, a seed, apollen, an anther and a calyx and the like of the said plant or culturedcells which are obtained by the tissue culture thereof in theabove-mentioned medium or another conventional medium can be used.

The present invention can also be applied to neoplastic cell and/orhairy-root, obtained by infecting a plant tissue with Agrobacteriumtumefaciens or Agrobacterium rhizogenes.

By culturing these tissues or cells in the presence of at least onesubstance selected from the group consisting of jasmonic acids,compounds containing a heavy metal, complex ions containing a heavymetal, heavy metal ions, amines, and antiethylene agents, culturedtissues or cultured cells having higher taxane-type diterpeneproductivity than that of those obtained by the tissue culture carriedout under the normal culture conditions, can be obtained.

When at least one compound selected from compounds containing a heavymetal, complex ions containing a heavy metal, heavy metal ions, amines,and antiethylene agents is used together with jasmonic acids representedby the above-mentioned general formulae (I), (II) or (III), the effectof the first inventiont of the present application can be enhanced.

Taxane-type diterpene can be fractionated from the cultures such ascultured tissues, cultured cells and culture medium, which are obtainedaccording to the above-mentioned process, by extraction with an organicsolvent such as methanol. It is also possible to recover the taxane-typediterpene continuously during culture by allowing an appropriateadsorbing agent or an organic solvent coexist in the culture medium.

One preferable example of the tissue culture according to the presentinvention can be illustrated as follows.

A piece of a plant body of a plant belonging to genus Taxus, such as aroot, a growing point, a leaf, a stem, a seed and the like is sterilizedand placed on Woody Plant Medium solidified with gelan gum, and kept at10-35° C. for about 14-60 days so that a part of the tissue piece ischanged to callus. By subculturing the callus thus obtained, the growingspeed is gradually increased and stabilized callus can be obtained. Bythe stabilized callus, we refer to a callus which remains in callusstate during culture without showing differentiation into a shoot or aroot and the cells of which have uniform growing speed.

Such stabilized callus is transferred to a liquid medium, suited for thegrowth, such as liquid Woody Plant Medium and grown. The growing speedis further increased in the liquid medium. According to the presentinvention, the stabilized callus or the cells constituting theabove-mentioned callus are grown in a solid medium or a liquid medium inthe presence of at least one substance selected from a group consistingof jasmonic acids, compounds containing a heavy metal, complex ionscontaining a heavy metal, heavy metal ions, amines and antiethyleneagents. And, it is also possible to fractionate the stabilized callus orthe cells constituting the said callus into a plurality of layersaccording to the difference in their specific gravities and grow thecells contained in at least one layer in a culture medium containing atleast one substance selected from the group consisting of jasmonicacids, compounds containing a heavy metal, complex ions containing aheavy metal, heavy metal ions, amines and antiethylene agents.

In a generally known method to fractionate the cells according to theirspecific gravities, density gradient is formed by a medium forcentrifugal separation, and the cells are layered over it, thencentrifugal separation is carried out.

As a medium for centrifugal separation, Ficoll, Percoll (both producedby Pharmacia LKB Biotechnology Co. Ltd.,), sucrose and cesium chlorideand the like are used. In the examples including Example No. 5, thedensity gradient was produced by the use of Ficoll, however, the mediumis not particularly limited to any substance as far as it does notdamage the cells.

The number of the layers forming the density gradient is notparticularly restricted. The difference between the specific gravitiesof layers is not particularly limited and each difference in thespecific gravity can be the same or different.

Accordingly, the definition of the density gradient includes a casewherein the gradient changes continuously (the condition wherein thenumber of the layers forming the density gradient is close to infinite,and the specific gravity difference between each layer is close to 0).

The cells can be fractionated into a plurality of layers according tothe difference in their specific gravities by thus forming the densitygradient, layering the cells and carrying out the centrifugalseparation.

The specific gravity of the layer to be formed is normally in the rangeof 1.00 to 1.20 g/ml, preferably in the range of 1.03 to 1.11 g/ml. As alayer to become an object for culture, at least one layer is selected,but it is also possible to select all the layers and culture them.

When a plurality of layers are selected and cells contained in theselected layers are cultured, it is possible to culture the cells inthese layers individually, but, it is also possible to mix the cells intwo or more layers of the selected plurality of layers and culture them.

The cultured cells having high taxane-type diterpene productivity can beusually obtained by culturing cells contained in a layer having thespecific gravity of 1.07 or less, but it is not always limited to thisrange, since it may fluctuate depending on the cells to be cultured orthe culture conditions. There is also a tendency that the cells in alayer of a higher specific gravity, have a higher content of thetaxane-type diterpene at the time when the fractionation is carried outaccording to the difference in the specific gravities. Accordingly, toensure that cultured cells which produce the taxane-type diterpene at ahigh rate can be obtained, it is desirable that the cells in all thefractionated layers are cultured for a certain period, then theconcentration of the taxane-type diterpene in the cells of each layer ismeasured, and the layer containing the cultured cells which produce thetaxane-type diterpene at a high rate is selected from among thoselayers.

It is also possible to fractionate the cultured cells into a pluralityof layers according to the difference in the specific gravities bypreparing a medium for centrifugal separation having one particularspecific gravity such as 1.07 g/ml, for example, and carrying out thecentrifugal separation according to the above-mentioned method.

Furthermore, the first invention of the present application can be usedtogether with the method of the second invention of the presentapplication wherein the culture is carried out by controlling the oxygenconcentration in a gas phase in a culture vessel to less than the oxygenconcentration in the atmosphere, from the initial stage of the culture,or by controlling the dissolved oxygen concentration in a fluid mediumwhich is in contact with the tissue or the cell to less than thesaturated dissolved oxygen concentration at that temperature from theinitial stage of the culture.

Here, by the initial stage of the culture, we refer to from the timewhen the culture was started through the 7th day after the start of theculture, and the controlling of the oxygen concentration in the gasphase in the culture vessel or the controlling of the dissolved oxygenconcentration in the fluid medium which is in contact with the tissue orthe cell is preferably done from the beginning of the culture. Thecontrolling period is not particularly limited, and, the controllingunder the said conditions can be done in the entire culture period, oronly in a part of the entire culture period, however, it is preferableto carry out the control at least for 3 days during the entire cultureperiod.

The oxygen concentration in the gas phase in the culture vessel isrequired to be controlled to 4-15%, and it is particularly preferable tocontrol it to 6-12%. The dissolved oxygen concentration in the fluidmedium is required to be controlled to 1-75% of the saturated dissolvedoxygen concentration at that temperature and it is particularlypreferable to control it to 10-75%.

It is also possible to combine the first invention of the presentapplication, the second invention of the present application and thethird invention of the present application all together.

According to the first invention of the present application, it iseffective to add jasmonic acids when the cultured cells are in theexponential growth phase or in the stationary phase, and it isparticularly preferable to add jasmonic acids in a transitional periodfrom the exponential growth phase to the stationary phase. The same canbe said of the timing of the treatment for increasing the amount of theendogenous jasmonic acids to be produced. For example, when cells aresubcultured in every 21 days, the 7th-16th day is the suitable time foraddition of the jasmonic acids or the treatment to increase the amountof the endogenous jasmonic acids to be produced, and when the cells inthe exponential growth phase, for example those on the 7th-14th day areto be subcultured, the suitable time is immediately after thetransplantation. The addition of the jasmonic acids or the treatment toincrease the amount of the endogenous jasmonic acid to be produced canbe done at a time, in a plurality of parts, or continuously.

It is effective to add compounds containing a heavy metal, complex ionscontaining a heavy metal or heavy metal ions after the beginning of theculture and before the transitional period of the cultured cells fromthe exponential growth phase to the stationary phase, and it isparticularly preferable to add them at the beginning of the culture. Theaddition of the said compounds or the ions can be done at a time, or ina plurality of parts.

It is effective to add amines before the transitional period of thecells from the exponential growth phase to the stationary phase, and itis particularly preferable to add them at the beginning of the culture.The addition of said compounds can be done at a time or in a pluralityof parts.

It is effective to add antiethylene agents before the transitionalperiod of the cells from the exponential growth phase to the stationaryphase, and it is particularly preferable to add them immediately afterthe transition to the stationary phase. The addition of the saidcompounds can be done at a time or in a plurality of parts.

The temperature for the tissue culture according to the first inventionof the present application is usually about 10-about 35° C., andpreferably about 23-28° C. according to the high growing speed. As forthe culture period, 14-42 days are preferable.

When a liquid medium is used for the culture according to the firstinvention of the present application, the cultured cells can befractionated from the culture medium after the culture is completed, bysuch a method as decantation or filtration and the desired taxane-typediterpene can be fractionated from the cultured cells and/or the culturemedium by such a method as extraction with an organic solvent .

The second invention of the present application will be explained asfollows.

According to the second invention of the present application, theculture of the plant means culture of a tissue or a cell of the plant,wherein the culture is carried out by a conventionally known processexcept that the culture is carried out by controlling the oxygenconcentration in the gas phase of the culture vessel to below theatmospheric oxygen concentration from the initial stage of the culture,or by controlling the dissolved oxygen concentration in the fluid mediumwhich is in contact with the tissue or the cell to below the saturateddissolved oxygen concentration at that temperature from the initialstage of the culture.

So far, in the culture of a plant producing the taxane-type diterpene,there has been no reports wherein the culture is carried out under suchconditions that the oxygen concentration in the gas phase to be suppliedto the culture vessel wherein the tissue or the cells are cultured orthe dissolved oxygen concentration in the medium which is in contactwith the tissue or the cells to below the atmospheric oxygenconcentration or below the saturated dissolved oxygen concentration, andit was beyond all expectations that the amount of the taxane-typediterpene to be produced is increased by that.

According to the second invention of the present application, the oxygenconcentration in the gas phase of the culture vessel wherein the tissueor the cells are cultured is required to be controlled to 4-15%, it isparticularly preferably controlled to 6-12%. The dissolved oxygenconcentration of the fluid medium which is in contact with the tissue orthe cells is required to be controlled to 1-75% of the saturateddissolved oxygen concentration at that temperature, it is particularlypreferably controlled to 10-75%.

Examples of a medium to be used in the second invention of the presentapplication, include the medium conventionally known for the tissueculture of a plant, such as medium of Murashige & Skoog (1962), mediumof Linsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg's B-5medium, and Mitsui's M-9 medium and the like.

A phytohormone, and if necessary a carbon source, an inorganiccomponent, vitamins, amino acids and the like may be added as well tothese media.

As a carbon source, a disaccharide such as sucrose, maltose, andlactose, a monosaccharide such as glucose, fructose and galactose,starch or a mixture of two or more kinds of such sugar sources mixed atan appropriate ratio can be utilized.

As an inorganic component, illustrative examples include phosphorus,nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc,boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, andthese components can be added in the form of such a compound aspotassium nitrate, sodium nitrate, calcium nitrate, potassium chloride,potassium monohydrogenphosphate, potassium dihydrogenphosphate, calciumchloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferricsulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate,sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chlorideand the like.

As the phytohormone, for example, auxin such as indoleacetic acid (IAA),naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid(2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can beused.

As the vitamins, for example, biotin, thiamin (vitamin B₁), pyridoxine(vitamin B₆), pantothenic acid, inositol, nicotinic acid and the likecan be used.

As the amino acids, for example, glycine, phenylalanine, leucine,glutamine, cysteine and the like can be added.

Generally, the carbon source in a concentration of about 1-about 30 g/l,the inorganic component in a concentration of about 0.1 μM-about 100 mM,the phytohormones in a concentration of about 0.01-about 10 μM, and thevitamins and the amino acids respectively in a concentration of about0.1-about 100 mg/l are used.

According to the second invention of the present application, both aliquid medium and such a solid medium that contains agar and gelan gumnormally in an amount of 0.1-1% can be used.

According to the tissue culture of the second invention of the presentapplication, a piece of a tissue or a cell of a root, a growing point, aleaf, a stem, a seed, a pollen, an anther and a calyx and the like ofthe said plant or cultured cells which are obtained by the tissueculture thereof in the above-mentioned medium or another conventionalmedium can be used.

The second invention of the present application can also be applied toneoplastic cell and/or hairy-root, obtained by infection withAgrobacterium tumefaciens or Agrobacterium rhizogenes.

When these tissues or cells are cultured by controlling the oxygenconcentration in the gas phase in the culture vessel to less than theoxygen concentration in the atmosphere, from the initial stage of theculture, or by controlling the dissolved oxygen concentration in thefluid medium which is in contact with the tissue or the cell to lessthan the saturated dissolved oxygen concentration at that temperature,from the initial stage of the culture, cultured tissue or the culturedcells having higher taxane-type diterpene productivity than that ofthose obtained by the tissue culture carried out under normal cultureconditions can be obtained.

According to the second invention of the present application, theinitial stage of the culture refers to from the time when the culturewas started through the 7th day after the start of the culture, and thecontrolling of the oxygen concentration in the gas phase in the culturevessel or the controlling of the dissolved oxygen concentration in thefluid medium which is in contact with the tissue or the cell ispreferably done from the beginning of the culture.

The controlling period is not particularly limited, and, the controllingunder the said condition can be done in the entire culture period, oronly in a part of the entire culture period, however, it is preferableto carry out the control at least for 3 days during the entire cultureperiod.

The production method according to the second invention of the presentapplication can be used together with a culture method carried out inthe presence of various kinds of taxane-type diterpene productionpromoting substances to further increase the productivity of thetaxane-type diterpene.

Examples of the taxane-type diterpene production promoting substanceinclude, for example, jasmonic acids represented by the above-mentionedgeneral formulae (I), (II) or (III), compounds containing a heavy metal,complex ions containing a heavy metal, heavy metal ions, amines andantiethylene agents to be used for the above-mentioned first inventionof the present application.

Also the second invention of the present application can be also usedtogether with the method of the third invention of the presentapplication which will be described later in detail, wherein the cellsare fractionated into a plurality of layers according to the differencein their specific gravities, and the cells contained in at least onelayer are cultured.

The production method according to the second invention of the presentapplication can be used together with both the said method according tothe first invention of the present application wherein culture iscarried out in the presence of the jasmonic acids and the like and themethod according to the third invention of the present applicationwherein the cells are fractionated into a plurality of layers accordingto the difference in their specific gravities and cells contained in atleast one layer are cultured.

Taxane-type diterpene can be fractionated from the cultures such ascultured tissues, cultured cells and culture medium, which are obtainedaccording to the above-mentioned process, by extraction with an organicsolvent such as methanol.

One preferable example of the tissue culture according to the secondinvention of the present application can be illustrated as follows.

A piece of a plant body of a plant belonging to genus Taxus, such as aroot, a growing point, a leaf, a stem, a seed and the like is sterilizedand placed on Woody Plant Medium solidified with gelan gum, and kept at10-35° C. for about 14-60 days so that a part of the tissue piece ischanged to callus. By subculturing the callus thus obtained, the growingspeed is gradually increased and stabilized callus can be obtained. Bythe stabilized callus, we refer to a callus which remains in callusstate during culture without showing differentiation into a shoot or aroot and the cells of which have uniform growing speed.

Such stabilized callus is transferred to a liquid medium, suited for thegrowth, such as liquid Woody Plant Medium and grown. The growing speedis further increased in the liquid medium. According to the presentinvention, the stabilized callus or the cells constituting theabove-mentioned callus is grown under the culture conditions wherein theoxygen concentration in a gas phase in a culture vessel is controlled toless than the oxygen concentration in the atmosphere from the initialstage of the culture, or the dissolved oxygen concentration in a fluidmedium which is in contact with the tissue or the cell is controlled toless than the saturated dissolved oxygen concentration at thattemperature, from the initial stage of the culture.

The tissue or the cell gains energy necessary for maintenance and growthof individium, by consuming oxygen (respiration). It is generally knownthat when a tissue or a cell is cultured, the cell mass is increased andthe amount of the oxygen consumption is increased as well with thepassage of culture period. Accordingly unless a ventilation gas isforcedly supplied from outside of the system, the oxygen concentrationin the gas phase in the culture vessel such as a flask wherein thetissue or the cell is contained, or the dissolved oxygen concentrationin the medium which is in contact with the tissue or the cell naturallydecreases to a value less than the oxygen concentration in theatmosphere, or the saturated dissolved oxygen concentration at thattemperature, with the passage of culture period.

The present invention is different from the above-mentioned finding onthe point that the culture is carried out by actively controlling theoxygen concentration in the gas phase in the culture vessel whichcontains the tissue or the cell or the dissolved oxygen concentration inthe culture medium to less than the oxygen concentration in theatmosphere or the saturated dissolved oxygen concentration at thattemperature.

In one illustrative process to enhance the effect of the presentinvention, the oxygen concentration in the gas phase in the culturevessel or the dissolved oxygen concentration in the fluid medium ispreviously controlled to less than the oxygen concentration in theatmosphere or the saturated dissolved oxygen concentration at thattemperature, prior to the subculture of the tissue or the cell in theculture vessel.

The controlling period is not particularly limited as mentioned above,however, it is preferable to carry out the control at least for 3 daysduring the entire culture period.

In addition to that, the controlling method is not particularly limitedto any as far as it is a method wherein the oxygen concentration in thegas phase in the culture vessel which contains the tissue or the cell,or the dissolved oxygen concentration in the fluid medium which is incontact with the tissue or the cell, can be controlled to less than theoxygen concentration in the atmosphere or the saturated dissolved oxygenconcentration at that temperature, and in some examples of such method,a gas having a controlled oxygen concentration, which is obtained bymixing an air with nitrogen and the like to lower the oxygenconcentration, is directly sent into the gas phase in the culture vesselor the culture medium, or such a gas is directly sent into the culturemedium outside of the culture vessel, i.e. in an aeration tank and thelike, then the culture medium is poured into the culture vessel, or agas such as air to be supplied to the culture vessel is directly sentinto the gas phase or the culture medium by controlling the feed speed,or such a gas is directly sent into the culture medium outside of theculture vessel, i.e. in an aeration tank and the like then the culturemedium is poured into the culture vessel, or the culture vessel isplaced under low oxygen atmosphere to carry out culture or the cultureis carried out in the presence of an oxygen adsorbing agent.

The temperature for the tissue culture according to the presentinvention is usually about 10-about 35° C., and preferably about 23-28°C. according to the high growing speed. As for the culture period, 14-42days are preferable.

When a liquid medium is used for the culture according to the presentinvention, the cultured cells can be fractionated from the culturemedium after the culture is completed by such a method as decantation orfiltration and the desired taxane-type diterpene can be fractionatedfrom the cultured cells and/or the culture medium by such a method asextraction with an organic solvent. It is also possible to recover thedesired compound continuously during the culture by allowing anadsorbing agent or an appropriate organic solvent coexist in the culturesystem.

The third invention of the present application will be explained asfollows.

According to the third invention of the present application, a layercontaining the cultured cells, which shall show high taxane-typediterpene productivity after being cultured, can be exemplified by alayer having the specific gravity of 1.07 or less.

In a generally known method to fractionate the cells according to theirspecific gravities, a density gradient is formed by a medium forcentrifugal separation, and the cells are layered over it, thencentrifugal separation is carried out.

As a medium for centrifugal separation, Ficoll, Percoll (both producedby Pharmacia LKB Biotechnology Co. Ltd.,), sucrose and cesium chlorideand the like are used. In Examples, the density gradient was produced bythe use of Ficoll, however, the medium is not particularly limited toany substance as far as it does not damage the cells. Ficoll has beenused for separation of cell granules and the like (Hess, R. et al.,Nature 208 (1965), 856-858) or separation of animal cells (Walder, I. A.et al., Proc. Soc. exptl. Biol. Med., 112(1963) 494-496) and the like.

The number of the layers forming the density gradient is notparticularly limited.

In Examples, a density gradient wherein the difference of the specificgravity between each layer is 0.02 is formed by the layers having thespecific gravity of 1.03, 1.05, 1.07, 1.09 and 1.11 (g/ml), however, thedifference of the specific gravity is not limited to this value, and thedifference of the specific gravity between each layer can be the same ordifferent.

Accordingly, the definition of the density gradient includes a casewherein the gradient changes continuously (the condition wherein thenumber of the layers forming the density gradient is close to infinite,and the difference of the specific gravity between each layer is closeto 0).

The cells can be fractionated into a plurality of layers according tothe difference in their specific gravities by thus forming the densitygradient, layering the cells and carrying out the centrifugalseparation.

The specific gravity of the layer to be formed is normally in the rangeof 1.00 to 1.20 g/ml, preferably in the range of 1.03 to 1.11 g/ml. As alayer to become an object for culture, at least one layer is selected,but it is also possible to select all the layers and culture them.

When a plurality of layers are selected and cells contained in theselected layers are cultured, it is possible to culture the cells inthese layers individually, but, it is also possible to mix the cells intwo or more layers of the selected plurality of layers and culture them.

The cultured cells having high taxane-type diterpene productivity can beusually obtained by culturing cells contained in a layer having thespecific gravity of 1.07 or less, but, it is not always limited to thisrange, since it may fluctuate depending on the cells to be cultured orthe culture conditions. There is also a tendency that the cells in alayer of a higher specific gravity, have a higher content of thetaxane-type diterpene at the time when the fractionation is carried outaccording to the difference in the specific gravities. Accordingly, toensure that cultured cells which produce the taxane-type diterpene at ahigh rate can be obtained, it is desirable that the cells in all thefractionated layers are cultured for a certain period, then theconcentration of the taxane-type diterpene in the cells of each layer ismeasured, and the layer containing the cultured cells which produce thetaxane-type diterpene at a high rate is selected from among thoselayers.

So far, there have been no cases reported wherein the cultured cells ofa plant producing the taxane-type diterpene are cultured after they arefractionated according to the specific gravity of the cells, and it wasbeyond all expectations that the cells can be fractionated into layersof cells each having different taxane-type diterpene productivity, bythe difference of the specific gravities, and that the cells whichproduce the taxane-type diterpene at a high rate can be obtained byculture of cells which are contained in a layer having the specificgravity of 1.07 or less, and whose taxane-type diterpene content is notso high at the time when they are fractionated.

According to the present invention, it is also possible to fractionatethe cultured cells into a plurality of layers according to thedifference in the specific gravities by preparing a medium forcentrifugal separation having one particular specific gravity such as1.07 g/ml, for example, and carrying out the centrifugal separationaccording to the above-mentioned method.

The culture medium to be used for the present invention includes typicalculture medium components. As such a component, an inorganic componentand a carbon source are typically used, and phytohormones, vitamins, andif necessary, amino acids can be added as well. As a carbon source, adisaccharide such as sucrose, maltose, and lactose, monosaccharide suchas glucose, fructose and galactose, starch or a mixture of two or morekinds of such sugar sources mixed at an appropriate ratio can beutilized.

As an inorganic component, illustrative examples include phosphorus,nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc,boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, andthese components can be added in the form of such a compound aspotassium nitrate, sodium nitrate, calcium nitrate, potassium chloride,potassium monohydrogenphosphate, potassium dihydrogenphosphate, calciumchloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferricsulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate,sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chlorideand the like.

As the phytohormone, for example, auxin such as indoleaceacid (IAA),naphthalenacetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D),and cytokinin such as kinetin, zeatin, dihydrozeatin can be used.

As the vitamins, for example, biotin, thiamin (vitamin B₁),pyridoxine(vitamin B₆), pantothenic acid, inositol, nicotinic acid andthe like can be used.

As the amino acids, for example, glycine, phenylalanine, leucine,glutamine, cysteine and the like can be added.

Generally, the inorganic component in a concentration of about 0.1μM-about 100 mM, the carbon source in a concentration of about 1-about30 g/l, the phytohormone in a concentration of about 0.01-about 10 μM,and the vitamins and the amino acids respectively in a concentration ofabout 0.1-about 100 mg/l are used.

Examples of a medium to be used for the present invention include thoseknown media which have been conventionally used for the plant tissueculture, such as Medium of Murashige & Skoog (1962), medium of LinsmaierSkoog (1965), Woody Plant Medium (1981), Gamborg's B-5 medium andMitsui's M-9 medium to which the above-mentioned phytohormone, and ifnecessary, the above-mentioned carbon source, vitamins and amino acidsare added.

According to the present invention, both a liquid medium and such asolid medium that contains agar and gelan gum normally in an amount of0.1-1% can be used, however, usually a liquid medium is preferable.

According to the tissue culture of the present invention, a piece of atissue or a cell of a root, a growing point, a leaf, a stem, a seed, apollen, an anther and a calyx and the like of the said plant, orcultured cells which are obtained by the tissue culture thereof in thesaid medium or another conventional medium can be used.

By fractionating these cells into particular specific gravity rangesthen culturing them according to the present invention, cultured cellshaving higher taxane-type diterpene productivity, in comparison withthose in the control area wherein no fractionation was carried out, canbe obtained. The taxane-type diterpene can be fractionated from thesecultured cells by extraction with an organic solvent such as methanol.

One preferable example of the tissue culture according to the presentinvention can be illustrated as follows.

A piece of a plant body of a plant belonging to genus Taxus, such as aroot, a growing point, a leaf, a stem, a seed and the like is sterilizedand placed on Woody Plant Medium solidified with gelan gum, and kept at10-35° C. for 14-60 days so that a part of the tissue piece is changedto callus. By subculturing the callus thus obtained, the growing speedis gradually increased and stabilized callus can be obtained. By thestabilized callus, we refer to a callus which remains in callus stateduring culture without showing differentiation into a shoot or a rootand the cells of which have uniform growing speed.

Such stabilized callus is transferred to a liquid medium, suited for thegrowth, such as liquid Woody Plant Medium and grown. The growing speedis further increased in the liquid medium.

The temperature for the tissue culture according to the presentinvention is usually about 10-about 35° C., and preferably about 23-28°C. according to the high growing speed. As for the culture period, 14-42days are preferable.

When a liquid medium is used for the culture according to the presentinvention, the cultured cells can be fractionated from the culturemedium after the culture is completed, by such a method as decantationor filtration and the desired taxane-type diterpene can be fractionatedfrom this by such a method as extraction with an organic solvent.

According to the first invention and the second invention of the presentapplication, taxane-type diterpene can be easily obtained in largequantity.

According to the third invention of the present application, culturedcells which produce taxane-type diterpene at a high rate can be obtainedwith a simple operation.

When the first, second or third invention of the present application isto be industrially executed, the efficiency can be further increased byemploying the following fourth, fifth, sixth or seventh invention of thepresent application in an independent form or in a combined form.

That means, it is necessary to supply a gas containing oxygen to aculture liquid to culture tissues or cells of a plant which producestaxane-type diterpene. Normally, air is used for this purpose, however,after an intensive study, the present inventors found that thetaxane-type diterpene production can be efficiently carried out by theuse of a gas containing 0.03-10%, preferably 0.1-5% of carbon dioxide,as a gas to be introduced to a tank for culturing the tissues or thecells of the plant producing the taxane-type diterpene, and completedthe fourth invention of the present application.

The present inventors also found that the productivity of thetaxane-type diterpene in the cultures can be remarkably improved and thefluctuation of the taxane-type diterpene productivity due to thesubculture can be controlled by carrying out a two-stage culture of thetissue or the cell of the plant producing the taxane-type diterpene,comprising a first stage using a medium to which an oxidizing agent or awater soluble organic compound containing oxygen is added for obtainingthe tissues or the cells which is activated for production of thetaxane-type diterpene in the subsequent stage, and a second stage whichis carried out such conditions that promote the production of thetaxane-type diterpene, and completed the fifth invention of the presentapplication. Here, examples of the oxidizing agent includeperoxodisulfates such as potassium peroxodisulfate and hydrogenperoxide, and examples of the water soluble organic compound containingoxygen include dimethyl formamide, dimethyl sulfoxide, and ethyleneglycol and the like. The total concentration of the above-mentionedadditive in the culture medium is preferably 10⁻⁶ M-10⁻¹ M immediatelyafter the addition, and it is further preferable to control theconcentration to be in the range of 10⁻⁵ M to 10⁻² M.

The present inventors also found that the high density culture of thetissue or the cell of the plant producing the taxane-type diterpene canbe carried out by inoculating the tissues or the cells in a culturemedium containing a saccharide in a concentration of 2-50 g/l,preferably 10-30 g/l, and/or nitrate ion in a concentration of 2-50mmol/l, preferably 10-30 mmol/l, then by adding a nutrient sourcesolution containing the saccharide in an amount of 0.2-5 g/l, preferably0.5-3 g/l, and/or nitrate ion in an amount of 0.2-5 mmol/l, preferably0.5-3 mmol/l per day with respect to the initial volume of the saidculture medium, continuously or intermittently to the culture medium,thereby the taxane-type diterpene production volume per culture vesselcan be remarkably increased and completed the sixth invention of thepresent application. Here, by the density, we refer to a cell mass pervolume of the culture solution in the culture vessel, which is shown interms of dry cell mass (g) per liter of the culture solution. Accordingto the sixth invention of the present application, it is preferable tocarry out culture while the culture medium is renewed by adding thenutrient source solution and simultaneously separating and removing thesame volume of the medium from the tissues or the cells and to recoverthe taxane-type diterpene from at least one selected from the resultingcultures, the medium recovered by removal during the culture, and themedium obtained at the end of the culture. The sixth invention of thepresent application is particularly effective in improving thetaxane-type diterpene productivity in the high density culture whereinthe density of the tissue or the cell of the above-mentioned plant atthe start of the culture with respect to the medium volume is 50 g freshweight/l or higher.

Furthermore, though the culture is normally finished when the cells ofhigh density are obtained, the present inventors achieved, through theintensive study, the continuous culture by continuing the culture whilethe cells are removed, and after further examination, finally completeda continuous culture method, which is the seventh invention of thepresent application. That means, the taxane-type diterpene can beproduced with such a high rate that could be hardly attained with theconventional process, by adding the fresh medium continuously orintermittently in such a way that the specific renewing ratio defined bythe dimensionless number F=V_(I) /V/μ (wherein, V is the total volume ofthe culture medium in a culture tank, V_(I) is the feed speed of thefresh medium, and μ is the specific growth rate of the tissues or thecells) is in the range of 0.1 to 10, and by recovering the taxane-typediterpene from the culture medium containing the tissues or the cellswhich is continuously or intermittently taken out from the tank and/orthe culture solution which does not contain the tissue nor the cell andwhich is continuously or intermittently taken out from the tank, andcompleted the seventh invention of the present application. It isfurther preferable to set the specific renewing ratio of the culturemedium, F, to 0.5-5. The saccharide concentration in the culturesolution is preferably 5-40 g/l, and the nitrate ion concentration inthe culture solution is preferably 10-40 mmol/l. The present inventioncan be effective with the cell density in terms of fresh cell weight perlitter of 50-500 g, however, the higher the density is as far as it isin a range wherein extremely vigorous stirring is not required, the moreefficiently the taxane-type diterpene can be produced, thus thepreferable density 200 g or higher per liter.

In order to combine the above-mentioned fourth, fifth, sixth, or seventhinvention of the present application with the above-mentioned thirdinvention of the present application, the cells obtained according tothe third invention of the present application can be cultured accordingto the fourth, fifth, sixth or seventh invention of the presentapplication to produce the desired taxane-type diterpene.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. is a graph showing the change of the yield of the taxol in theculture medium after adding 100 μM of methyl jasmonate.

FIG. 2 is a graph showing the change of the yield of the baccatin IIIthe culture medium after adding 100 μM of methyl jasmonate.

FIG,. 3 is a diagram illustrating an example of a culture apparatus usedfor carrying out the tissue culture according to the second invention ofthe present application. Each numeral used in the FIG. 3 has thefollowing meaning.

1 Air feed pipe

2 Nitrogen feed pipe

3 Culture vessel

4 Sparger for supplying oxygenic gas

5 Electrode for dissolved oxygen

6 Dissolved oxygen concentration controller

7 Vent

8 Valve

9 Oxygen flow control valve

10 Air filter

11 Impeller

FIG. 4 is a graph showing the growth in the culture after thefractionation.

FIG. 5 is a graph showing the taxane content in the culture after thefractionation.

FIG. 6 is a graph showing the distribution of the cells at thefractionation.

FIG. 7 is a graph showing the taxane content (in cell) at thefractionation.

FIG. 8 is a diagram illustrating an example of a culture apparatus usedfor carrying out the tissue culture according to the sixth or theseventh invention of the present application. Each numeral and alphabetused in the FIG. 8 has the following meaning.

12 Medium feed pipe

13 Medium feed opening

14 Opening with a filter for taking out culture medium alone (theculture medium containing no tissues nor cells)

15 Culture medium outlet pipe

16 Sparger for supplying oxygenic gas

17 Impeller

18 Culture mixture (the culture solution containing tissues or cells)discharge pipe

19 Pressurized fluid inlet a, b, c, d and e Valves

BEST MODE FOR CARRYING OUT THE INVENTION

The invention will be further illustrated with the following examplesand comparative examples, however, these examples are not to beconstrued to limit the scope of the invention.

EXAMPLE 1

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide callus of Taxus baccata LINN. One gram(fresh weight) of the callus was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium to which theabove-mentioned component was added to give the same concentration andshake culture was carried out with a rotary shaker (amplification of 25mm, 120 rpm) and the callus was subcultured in every 21 days toaccelerate the growth rate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and shake culture was carried out at 25° C. for 14 days.On the 14th day after starting the culture, methyl ester of tuberonicacid (which is a compound represented by the general formula (I),wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,and R^(6a) are hydrogen atoms, R^(6b) is hydroxyl group, R⁷ is methoxygroup, n is 1 and C³ and C⁴ have a double bond between them) was addedas the compound represented by the general formula (I) to give the finalconcentration of 0.01-1000 μM, and the culture was further carried outfor another 7 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the yield of the cultured cells per litre of theliquid medium. Taxane-type diterpenes were extracted from the driedcallus with methanol and the like, and they were determined by comparingwith standard taxol, cephalomannine, and baccatin III using highperformance liquid chromatography to measure the yields of thetaxane-type diterpenes. The results are shown in Table 1.

COMPARATIVE EXAMPLE 1

The procedure of Example 1 was carried out except that the methyl esterof tuberonic acid was not added. The results are shown in Table 1.

EXAMPLE 2

The procedure of Example 1 was carried out except that the methyl esterof tuberonic acid was added successively a total of 4 times in every twodays starting from the 7th day after starting the culture (to give thefinal concentration of 25 μM each time, and to give the totalconcentration of 100 μM). The results are shown in Table 1.

EXAMPLE 3

The procedure of Example 1 was carried out except that 100 μM of themethyl ester of tuberonic acid was added on the first day after startingthe culture then the culture was further carried out for another 20days. The results are shown in Table 1.

EXAMPLE 4

The procedure of Example 1 was carried out except that 100 μM of themethyl ester of tuberonic acid was added on the 7th day after startingthe culture and the culture was further carried out for another 14 days.The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              tuberonate yield III taxol mannine                                            (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          14.3    0.4    3.5   1.2                                     Example 1                                                                     Example 1 0.01 14.3 0.4 4.6 1.3                                               " 0.1 13.5 0.5 5.5 1.5                                                        " 1 13.2 0.7 6.9 1.8                                                          " 10 13.0 0.9 14.8 2.2                                                        " 100 12.7 12.2 16.5 2.4                                                      " 250 12.5 14.5 21.1 2.2                                                      " 500 11.6 15.0 10.6 2.3                                                      " 1000 7.5 1.2 3.9 1.8                                                        Example 2 100 (25 × 4) 12.9 13.5 22.5 2.3                               Example 3 100 7.1 1.0 4.0 1.3                                                 Example 4 100 12.5 10.5 15.6 2.1                                            ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 5

The cells with accelerated growth rate obtained by the method of Example1 fractionated firstly by a stainless steel mesh and cell clustershaving the size of 250-840 μm were obtained. A medium having a specificgravity of 1.07 (g/ml) was produced by the use of Ficoll and theabove-mentioned cells were layered over it and centrifuged at 700 rpmfor 6 minutes. The cells were fractionated into two layers according tothe difference of the specific gravity. The cells contained in the layerof 1.07 g/ml or less were fractionated and washed with 2% sucrosesolution three times or more, to wash off Ficoll. After the washing, 1 g(fresh weight) of the cells was transferred to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium and shake culture wascarried out at 25° C. for 14 days. On the 14th day after starting theculture, methyl ester of tuberonic acid was added to it to give thefinal concentration of 250 μM, and the culture was further carried outfor another 7 days. After completing the culture, the procedure ofExample 1 was carried out. The results are shown in Table 2. Theproductivity of the taxane-type diterpenes could be greatly improved bythe combination of the selection of cells having a particular specificgravity and the addition of the methyl ester of tuberonic acid.

COMPARATIVE EXAMPLE 2

The procedure of Example 5 was carried out except that the methyl esterof tuberonic acid was not added. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              tuberonate yield III taxol mannine                                            (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          14.2    0.5    5.4   1.5                                     Example 2                                                                     Example 5 250 12.2 17.4 28.3 3.1                                            ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 6

250 μM of methyl ester of tuberonic acid was added to cultured cells ofTaxus brevifolia NUTT obtained on the 14th day after starting theculture by the method of Example 1, and the culture was further carriedout for another 7 days. After completing the culture, the procedure ofExample 1 was carried out. The results are shown in Table 3.

COMPARATIVE EXAMPLE 3

The procedure of Example 6 was carried out except that methyl ester oftuberonic acid was not added. The results are shown in Table 3.

EXAMPLE 7

The procedure of Example 6 was carried out except that cultured cells ofT. media were used. The results are shown in Table 3.

COMPARATIVE EXAMPLE 4

The procedure of Example 7 was carried out except that the methyl esterof tuberonic acid was not added. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              tuberonate yield III taxol mannine                                            (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          12.5    0.1    0.2   0.2                                     Example 3                                                                     Example 6 250 10.0 1.4 3.3 0.5                                                Comparative 0 13.6 0.2 0.3 0.1                                                Example 4                                                                     Example 7 250 9.5 10.4 5.4 0.5                                              ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 8

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide callus of Taxus baccata LINN. One gram(fresh weight) of the callus was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium to which theabove-mentioned component was added to give the same concentration andshake culture was carried out with a rotary shaker (amplification of 25mm, 120 rpm) and the callus was subcultured in every 21 days toaccelerate the growth rate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and shake culture was carried out at 25° C. for 14 days.On the 14th day after starting the culture, methyl ester of cucurbicacid (which is a compound represented by the general formula (II)wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³ and C⁴have a double bond between them) was added as one of jasmonic acids togive the final concentration of 0.01-1000 μM, and the culture wasfurther carried out for another 7 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the yield of the cultured cells per litre of theliquid medium. Taxane-type diterpenes were extracted from the driedcallus with methanol and the like, and they were determined by comparingwith standard taxol, cephalomannine, and baccatin III using highperformance liquid chromatography to measure the yields of thetaxane-type diterpenes. The results are shown in Table 4.

COMPARATIVE EXAMPLE 5

The procedure of Example 8 was carried out, except that the methyl esterof cucurbic acid was not added. The results are shown in Table 4.

EXAMPLE 9

The procedure of Example 8 was carried out except that the methyl esterof cucurbic acid was added successively a total of 4 times in every twodays starting from the 7th day after starting the culture (to give thefinal concentration of 25 μM each time, and to give the totalconcentration of 100 μM). The results are shown in Table 4.

EXAMPLE 10

The procedure of Example 8 was carried out except that 100 μM of themethyl ester of cucurbic acid was added on the first day after startingthe culture then the culture was further carried out for another 20days. The results are shown in Table 4.

EXAMPLE 11

The procedure of Example 8 was carried out except that 100 μM of themethyl ester of cucurbic acid was added on the 7th day after startingthe culture and the culture was further carried out for another 14 days.The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              cucurbate yield III taxol mannine                                             (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          11.3    0.2    3.3   2.4                                     Example 5                                                                     Example 8 0.01 11.3 0.3 4.3 2.6                                               " 0.1 11.1 0.3 5.4 3.1                                                        " 1 10.4 0.3 6.7 3.3                                                          " 10 10.4 0.5 11.3 4.0                                                        " 100 9.8 7.2 15.7 4.4                                                        " 250 9.6 10.6 17.7 5.3                                                       " 500 9.3 12.0 13.1 3.0                                                       " 1000 5.7 1.2 4.2 2.8                                                        Example 9 100 (25 × 4) 10.5 14.6 18.2 4.1                                Example 10 100 6.1 0.8 3.9 2.8                                                Example 11 100 9.5 8.4 16.8 4.1                                            ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 12

The cells with accelerated growth rates obtained by the method ofExample 8 were fractionated firstly by a stainless steel mesh and cellclusters having the size of 250-840 μm were obtained. A medium having aspecific gravity of 1.07 (g/ml) was produced by the use of Ficoll andthe above-mentioned cells were layered over it and centrifuged at 700rpm for 6 minutes. The cells were fractionated into two layers accordingto the difference of the specific gravity. The cells contained in thelayer of 1.07 g/ml or less were fractionated and washed with 2% sucrosesolution three times or more, to wash off Ficoll. After the washing, 1 g(fresh weight) of the cells was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium and shake culture wascarried out at 25° C. for 14 days. On the 14th day after starting theculture, methyl ester of cucurbic acid was added to it to give the finalconcentration of 250 μM, and culture was further carried out for another7 days. After completing the culture, the procedure of Example 8 wascarried out. The results are shown in Table 5. The productivity of thetaxane-type diterpenes could be greatly improved by the combination ofthe selection of cells having a particular specific gravity and theaddition of the methyl ester of cucurbic acid.

COMPARATIVE EXAMPLE 6

The procedure of Example 12 was carried out except that the methyl esterof cucurbic acid was not added. The results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              cucurbate yield III taxol mannine                                             (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          11.5    0.3    4.3   2.8                                     Example 6                                                                     Example 12 250 9.7 22.2 29.7 3.8                                            ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 13

250 μM of methyl ester of cucurbic acid was added to cultured cells ofTaxus brevifolia NUTT obtained on the 14th day after starting theculture by the method of Example 8, and the culture was further carriedout for another 7 days. After completing the culture, the procedure ofExample 8 was carried out. The results are shown in Table 6.

COMPARATIVE EXAMPLE 7

The procedure of Example 13 was carried out except that methyl ester ofcucurbic acid was not added. The results are shown in Table 6.

EXAMPLE 14

The procedure of Example 13 was carried out except that cultured cellsof T. media were used. The results are shown in Table 6.

COMPARATIVE EXAMPLE 8

The procedure of Example 14 was carried out except that the methyl esterof cucurbic acid was not added. The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              cucurbate yield III taxol mannine                                             (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          12.5    0.1    0.2   0.2                                     Example 7                                                                     Example 13 250 11.0 1.2 1.3 0.3                                               Comparative 0 13.6 0.2 0.3 0.1                                                Example 8                                                                     Example 14 250 12.3 5.2 3.9 0.2                                             ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 15

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide callus of Taxus baccata LINN. One gram(fresh weight) of the callus was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium to which theabove-mentioned component was added to give the same concentration andshake culture was carried out with a rotary shaker (amplification of 25mm, 120 rpm) and the callus was subcultured in every 21 days toaccelerate the growth rate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and shake culture was carried out at 25° C. for 14 days.On the 14th day after starting the culture, methyl ester of jasmonicacid (which is a compound represented by the general formula (III)wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵,and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³ and C⁴have a double bond between them, 90% of which is in the trans-form and10% of which is in the cis-form) was added as one of jasmonic acids togive the final concentration of 0.01-1000 μM, and the culture wasfurther carried out for another 7 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the yield of the cultured cells per litre of theliquid medium. Taxane-type diterpenes were extracted from the driedcallus with methanol and the like, and they were determined by comparingwith standard taxol, cephalomannine, and baccatin III using highperformance liquid chromatography to measure the yields of thetaxane-type diterpenes. The results are shown in Table 7.

COMPARATIVE EXAMPLE 9

The procedure of Example 15 was carried out except that the methyl esterof jasmonic acid was not added. The results are shown in Table 7.

EXAMPLE 16

The procedure of Example 15 was carried out except that the methyl esterof jasmonic acid was added successively a total of 4 times in every twodays starting from the 7th day after starting the culture (to give thefinal concentration of 25 μM each time, and to give the totalconcentration of 100 μM). The results are shown in Table 7.

EXAMPLE 17

The procedure of Example 15 was carried out except that 100 μM of themethyl ester of jasmonic acid was added on the first day after startingthe culture then the culture was further carried out for another 20days. The results are shown in Table 7.

EXAMPLE 18

The procedure of Example 15 was carried out except that 100 μM of themethyl ester of jasmonic acid was added on the 7th day after startingthe culture and the culture was further carried out for another 14 days.The results are shown in Table 7.

EXAMPLE 19

The procedure of Example 15 was carried out except that jasmonic acid(which is a compound represented by the general formula (III) whereinR^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³, R⁴, R⁵, and R⁶are hydrogen atoms, R⁷ is hydroxy group, n is 1 and C³ and C⁴ have adouble bond between them; 90% of which is in the trans- form and 10% ofwhich is in the cis-form) was added as one of jasmonic acids to give thefinal concentration of 0.01-1000 μM. The results are shown in Table 8.

COMPARATIVE EXAMPLE 10

The procedure of Example 19 was carried out except that the methyl esterof jasmonic acid was not added. The results are shown in Table 8.

EXAMPLE 20

The analytical results of taxane-type diterpenes existed in the culturemedium of Example 15 prior to the addition of 100 μM of methyljasmonate, on the third day after the addition, and on the 7th day afterthe addition, are shown in FIG. 1 and FIG. 2 On the 7th day of theculture, about half of the taxol and about 70% of the baccatin III wereleaked in the medium.

COMPARATIVE EXAMPLE 11

The procedure of Example 20 was carried out except that the methyl esterof jasmonic acid was not added. The results are shown in FIG. 1 and FIG.2.

EXAMPLE 21

The cells with accelerated growth rate obtained by the method of Example15 were fractionated firstly by a stainless steel mesh and cell clustershaving the size of 250-840 μm were obtained. A medium having a specificgravity of 1.07 (g/ml) was produced by the use of Ficoll and theabove-mentioned cells were layered over it and centrifuged at 700 rpmfor 6 minutes. The cells were fractionated into two layers according tothe difference of the specific gravity. The cells contained in the layerof 1.07 g/ml or less were fractionated and washed with 2% sucrosesolution three times or more, to wash off Ficoll. After the washing, 1 g(fresh weight) of the cells was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium and shake culture wascarried out at 25° C. for 14 days. On the 14th day after starting theculture, methyl ester of jasmonic acid was added to it to give the finalconcentration of 250 μm, and the culture was further carried out foranother 7 days. After completing the culture, the procedure of Example15 was carried out. The results are shown in Table 9. The productivityof the taxane-type diterpenes could be greatly improved by thecombination of the selection of cells having a particular specificgravity and the addition of the methyl ester of jasmonic acid.

COMPARATIVE EXAMPLE 12

The procedure of Example 21 was carried out except that the methyl esterof jasmonic acid was not added. The results are shown in Table 9.

EXAMPLE 22

250 μM of methyl ester of jasmonic acid was added to cultured cells ofTaxus brevifolia NUTT obtained on the 14th day after starting theculture by the method of Example 15, and the culture was further carriedout for another 7 days. After completing the culture, the procedure ofExample 15 was carried out. The results are shown in Table 10.

Comparative Example 13

The procedure of Example 22 was carried out except that methyl ester ofjasmonic acid was not added. The results are shown in Table 10.

EXAMPLE 23

The procedure of Example 22 was carried out except that cultured cellsof T. media were used. The results are shown in Table 10.

COMPARATIVE EXAMPLE 14

The procedure of Example 23 was carried out except that the methyl esterof jasmonic acid was not added. The results are shown in Table 10.

                  TABLE 7                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              jasmonate yield III taxol mannine                                             (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          12.2    0.2    2.8   1.5                                     Example 9                                                                     Example 15 0.01 12.2 0.3 3.2 1.6                                              " 0.1 12.1 0.3 4.2 1.6                                                        " 1 11.3 0.4 4.9 1.8                                                          " 10 11.3 0.7 10.8 2.1                                                        " 50 10.9 3.1 10.9 2.2                                                        " 100 9.8 9.2 13.5 2.0                                                        " 250 10.0 12.9 15.1 2.0                                                      " 500 10.6 13.0 12.6 1.9                                                      " 1000 5.9 1.2 3.1 1.8                                                        Example 16 100 (25 × 4) 10.7 14.9 16.7 2.2                              Example 17 100 7.2 1.2 4.5 1.7                                                Example 18 100 9.7 10.6 14.2 2.1                                            ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

                  TABLE 8                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             jasmonic cell baccatin of cephalo-                                            acid yield III taxol mannine                                                  (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          12.2    0.2    1.2   0.4                                     Example 10                                                                    Example 19 0.01 12.2 0.3 2.2 0.6                                              " 0.1 12.2 0.3 3.4 0.6                                                        " 1 11.6 0.3 5.9 0.8                                                          " 10 11.5 0.5 8.3 2.6                                                         " 50 11.4 2.5 10.2 2.2                                                        " 100 10.3 7.2 12.8 3.0                                                       " 250 10.1 10.6 14.7 2.7                                                      " 500 10.2 12.0 11.1 1.1                                                      " 1000 6.7 1.2 1.2 0.5                                                      ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

                  TABLE 9                                                         ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              jasmonate yield III taxol mannine                                             (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          12.6    0.5    6.3   2.2                                     Example 12                                                                    Example 21 250 10.2 18.7 43.1 3.2                                           ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

                  TABLE 10                                                        ______________________________________                                        concentration          yield*)        yield*)                                   of  of yield*) of                                                             methyl cell baccatin of cephalo-                                              jasmonate yield III taxol mannine                                             (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0          12.5    0.1    0.2   0.2                                     Example 13                                                                    Example 22 250 10.2 3.4 4.3 0.5                                               Comparative 0 14.2 0.2 0.3 0.1                                                Example 14                                                                    Example 23 250 12.6 12.4 4.4 0.2                                            ______________________________________                                         *)The yield was calculated based on the total amount of production (in th     cell + in the medium).                                                   

EXAMPLE 24

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide callus of Taxus baccata LINN. One gram(fresh weight) of the callus was inoculated to an Erlenmeyer flaskcontaining 20 ml of liquid Woody Plant Medium to which theabove-mentioned component was added to give the same concentration andshake culture was carried out with a rotary shaker (amplification of 25mm, 100 rpm) and the callus was subcultured in every 21 days toaccelerate the growth rate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component was added to give the sameconcentration, and [Ag(S₂ O₃)₂ ]³⁻ was added to it as a compoundcontaining a heavy metal to give the final concentration of 10⁻⁹ M-1M.Then shake culture was carried out at 25° C. for 21 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the growth rate thereof. Taxane-type diterpenes wereextracted from the dried callus with methanol and the like, and theywere determined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpenes. The results are shown in Table 11.

EXAMPLE 25

The procedure of Example 24 was carried out except that [Ag(S₂ O₃)₂ ]³⁻was added on the 7th day after starting the culture to give the finalconcentration of 10-3M and the culture was further carried out foranother 14 days. After the completion of the culture, the procedure ofthe Example 24 was carried out. The results are shown in Table 11.

EXAMPLE 26

The procedure of Example 24 was carried out except that [Ag(S₂ O₃)₂ ]³⁻was added on the 14th day after starting the culture to give the finalconcentration of 10⁻³ M and the culture was further carried out foranother 7 days. After the completion of the culture, the procedure ofthe Example 24 was carried out. The results are shown in Table 11.

EXAMPLE 27

The procedure of Example 24 was carried out, except that [Ag(S₂ O₃)₂ ]³⁻was added on the 18th day after starting the culture to give the finalconcentration of 10⁻³ M and the culture was further carried out foranother 3 days. After the completion of the culture, the procedure ofExample 24 was carried out. The results are shown in Table 11.

EXAMPLE 28

The procedure of Example 24 was carried out, except that [Ag(S₂ O₃)₂ ]³⁻was added successively a total of 5 times at 4 days' intervals startingfrom the start (0 day) of the culture (to give the final concentrationof 2×10⁻⁴ M each time, and to give the total concentration of 10⁻³ M).The results are shown in Table 11.

EXAMPLE 29

The procedure of Example 24 was carried out except that methyl ester ofjasmonic acid (which is a compound represented by the general formula(III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³,R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³and C⁴ have a double bond between them) was added on the 14th day afterstarting the culture to give the final concentration of 10⁻⁴ M. Theresults are shown in Table 11.

EXAMPLE 30

The procedure of Example 24 was carried out except that the flask wasput in a vessel (the capacity of 3000 ml) having a gas feed opening anda gas discharge opening, then the vessel was closed hermetically, andair was so mixed with nitrogen that the concentration of the oxygen in agas to be supplied to the cells to be cultured became 10%, and the gaswas supplied through the feed opening at the rate of 25 ml per minute.The results are shown in Table 11.

EXAMPLE 31

The procedure of Example 30 was carried out except that methyl ester ofjasmonic acid was added on the 14th day of the culture to give the finalconcentration of 10⁻⁴ M. The results are shown in Table 11.

EXAMPLE 32

The procedure of Example 24 was carried out except that silver nitrateAg(NO₃) of 10⁻³ M was added at the start (0 day) of the culture insteadof [Ag(S₂ O₃)₂ ]³⁻. The results are shown in Table 11.

EXAMPLE 33

The procedure of Example 32 was carried out except that silver nitrateof 10⁻³ M was added on the 14th day of the culture. The results areshown in Table 11.

COMPARATIVE EXAMPLE 15

The procedure of Example 24 was carried out except that [Ag(S₂ O₃)₂ ]³⁻was not added. The results are shown in Table 11.

EXAMPLE 34

The procedure of Example 24 was carried out except that cobalt chloride(CoCl₂) was added instead of [Ag(S₂ O₃)₂ ]³⁻ as a compound containingthe heavy metal to give the final concentration of 10⁻⁹ M-1M. Theresults are shown in Table 12.

EXAMPLE 35

The procedure of Example 34 was carried out except that cobalt chloride(COCl₂) was added instead of [Ag(S₂ O₃)₂ ]³⁻ to give the finalconcentration of 10⁻⁵ M on the 7th day after starting the culture andthe culture was further carried out for another 14 days. Aftercompleting the culture, the procedure of Example 34 was carried out. Theresults are shown in Table 12.

EXAMPLE 36

The procedure of Example 34 was carried out except that cobalt chloridewas added to give the final concentration of 10⁻⁵ M on the 14th dayafter starting the culture and the culture was further carried out foranother 7 days. After completing the culture, the process of Example 34was carried out. The results are shown in Table 12.

EXAMPLE 37

The procedure of Example 34 was carried out except that cobalt chloridewas added to give the final concentration of 10⁻⁵ M on the 18th dayafter starting the culture, and the culture was further carried out foranother 3 days. After completing the culture, the procedure of Example34 was carried out. The results are shown in Table 12.

EXAMPLE 38

The procedure of Example 34 was carried out except that cobalt chloridewas added successively a total of 5 times at 4 days' intervals startingfrom the start (0 day) of the culture (to give the final concentrationof 2×10⁻⁶ M each time, and to give the total concentration of 10⁻⁵ M).The results are shown in Table 12.

EXAMPLE 39

The procedure of Example 34 was carried out except that methyl ester ofjasmonic acid (which is a compound represented by the general formula(III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³,R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³and C⁴ have a double bond between them) was added as one of jasmonicacids to give the final concentration of 10⁻⁴ M on the 14th day afterstarting the culture. The results are shown in Table 12.

EXAMPLE 40

The procedure of Example 34 was carried out except that the flask wasput in a vessel (the capacity of 3000 ml) having a gas feed opening anda gas discharge opening, then the vessel was closed hermetically, andair was so mixed with nitrogen that the concentration of the oxygen in agas to be supplied to the cells to be cultured became 10%, and the gaswas supplied through the feed opening at the rate of 25 ml per minute.The results are shown in Table 12.

EXAMPLE 41

The procedure of Example 40 was carried out except that methyl ester ofjasmonic acid was added to give the final concentration of 10⁻⁴ M on the14th day of the culture. The results are shown in Table 12.

                                      TABLE 11                                    __________________________________________________________________________    concentration                                                                              concentration                                                                        concentration                                                                         max oxygen        yield.sup.a) of                   of silver of silver of methyl concentration growth yield                                                                            .sup.a) of                                                                    cephalo- yield.sup                                                            .a) total                                                                     yield.sup.b)                                                                   thiosulfate                                                                  nitrate jasmonate                                                             in the gas rate                                                               baccatin III                                                                  mannine of taxol                                                              of taxane                                                                      (M) (M) (M)                                                                  phase (%) (times)                                                             (mg/l) (mg/l)                                                                 (mg/l) (mg/l)         __________________________________________________________________________    Comparative                                                                         0      0      0       20     2.7  0.14  0.56  0.28                                                                              0.98                    Example 15                                                                    Example 24 10.sup.-9 0 0 20 2.7 0.20 0.56 0.33 1.09                           " 10.sup.-8 0 0 20 2.8 0.29 0.97 0.76 2.02                                    " 10.sup.-7 0 0 20 2.7 0.34 2.47 1.57 4.38                                    " 10.sup.-6 0 0 20 2.6 1.06 2.86 1.80 5.72                                    " 10.sup.-5 0 0 20 2.7 1.13 4.10 7.95 13.18                                   " 10.sup.-4 0 0 20 2.7 7.62 5.11 14.53 27.26                                  " 10.sup.-3 0 0 20 2.7 27.90 5.96 25.13 58.99                                 " 10.sup.-2 0 0 20 2.3 0.72 5.50 5.80 12.02                                   " 10.sup.-1 0 0 20 1.9 0.26 2.15 1.37 3.78                                    " 1 0 0 20 1.2 0.08 0.36 0.20 0.64                                            Example 25 10.sup.-3 0 0 20 2.2 11.71 4.86 10.64 27.21                        Example 26 10.sup.-3 0 0 20 2.5 2.29 3.83 4.47 10.59                          Example 27 10.sup.-3 0 0 20 2.9 0.28 1.44 1.55 3.27                           Example 28 10.sup.-3 0 0 20 2.4 4.15 7.27 20.94 32.36                         Example 29 10.sup.-3 0 10.sup.-4 20 2.7 25.14 12.57 33.61 71.32                                                                      Example 30                                                                   10.sup.-3 0 0 10                                                              2.7 22.81 9.08                                                                27.45 59.34                                                                    Example 31                                                                   10.sup.-3 0                                                                   10.sup.-4 10 2.7                                                              27.00 12.34 34.73                                                             74.07                   Example 32 0 10.sup.-3 0 20 2.8 3.01 7.54 12.66 23.21                         Example 33 0 10.sup.-3 0 20 2.8 2.18 6.49 11.91 20.58                       __________________________________________________________________________     [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

                                      TABLE 12                                    __________________________________________________________________________           concentration                                                                         concentration                                                                         max oxygen          yield.sup.a) of                      of cobalt of methyl concentration growth yield.sup.a) of cephalo-                                                                  yield.sup.a) total                                                            yield.sup.b)                                                                   chloride jasmonate                                                            in the gas rate                                                              baccatin III                                                                  mannine of taxol                                                              of taxane                (M) (M) phase (%) (times) (mg/l) (mg/l) (mg/l) (mg/l)                       __________________________________________________________________________    Comparative                                                                          0       0       20      2.7  0.14   0.56   0.28 0.98                     Example 15                                                                    Example 34 10.sup.-9 0 20 2.5 0.15 0.56 0.27 0.98                             " 10.sup.-8 0 20 2.6 0.15 0.61 0.29 1.05                                      " 10.sup.-7 0 20 2.6 0.16 0.63 0.29 1.08                                      " 10.sup.-6 0 20 2.4 2.25 5.79 8.32 16.36                                     " 10.sup.-5 0 20 3.2 5.68 6.50 16.11 28.29                                    " 10.sup.-4 0 20 2.7 1.64 3.46 4.92 10.02                                     " 10.sup.-3 0 20 2.2 1.57 2.03 4.38 7.98                                      " 10.sup.-2 0 20 1.4 0.44 0.78 0.92 2.14                                      " 10.sup.-1 0 20 0.8 0.11 0.42 0.55 1.08                                      " 1 0 20 0.6 0 0 0 0                                                          Example 35 10.sup.-5 0 20 3.2 4.36 5.98 10.22 20.56                           Example 36 10.sup.-5 0 20 3.1 2.01 4.37 7.10 13.48                            Example 37 10.sup.-5 0 20 2.9 1.17 1.11 1.32 3.60                             Example 38 10.sup.-5 0 20 2.6 5.32 6.58 15.76 27.66                           Example 39 10.sup.-5 10.sup.-4 20 2.5 23.00 9.46 30.32 62.78                  Example 40 10.sup.-5 0 10 2.7 17.61 7.99 26.64 52.24                          Example 41 10.sup.-5 10.sup.-4 10 2.4 25.85 15.21 33.77 74.83               __________________________________________________________________________     [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

EXAMPLE 42

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent was added to give the same concentration and shake culture wascarried out with a rotary shaker (amplification of 25 mm, 100 rpm) andthe callus was subcultured in every 21 days to accelerate the growthrate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component had been added to give thesame concentration, then spermidine was added to it as amine to give thefinal concentration of 10⁻⁹ M-1M. Then shake culture was carried out at25° C. for 21 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the growth rate thereof. Taxane-type diterpenes wereextracted from the dried cells with methanol and the like, and they weredetermined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpene. The results are shown in Table 13.

EXAMPLE 43

The procedure of Example 42 was carried out except that spermidine wasadded to give the final concentration of 10⁻⁵ M on the 7th day afterstarting the culture and the culture was further continued for another14 days. After completing the culture, the procedure of Example 42 wascarried out. The results are shown in Table 13.

EXAMPLE 44

The procedure of Example 42 was carried out except that spermidine wasadded to give the final concentration of 10⁻ 1M on the 14th day afterstarting the culture and the culture was further continued for another 7days. After completing the culture, the procedure of Example 42 wascarried out. The results are shown in Table 13.

EXAMPLE 45

The procedure of Example 42 was carried out except that spermidine wasadded to give the final concentration of 10⁻⁵ M on the 18th day afterstarting the culture and the culture was further continued for another 3days. After completing the culture, the procedure of Example 42 wascarried out. The results are shown in Table 13.

EXAMPLE 46

The procedure of Example 42 was carried out except that spermidine wasadded successively a total of 5 times at 4 days' intervals starting fromthe start (0 day) of the culture (to give the final concentration of2×10⁻⁶ M each time, and to give the total concentration of 10⁻⁵ M). Theresults are shown in Table 13.

EXAMPLE 47

The procedure of Example 42 was carried out except that methyl ester ofjasmonic acid (which is a compound represented by the general formula(III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³,R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³and C⁴ have a double bond between them) was added as one of jasmonicacids on the 14th day after starting the culture to give the finalconcentration of 10⁻⁴ M. The results are shown in Table 13.

EXAMPLE 48

The procedure of Example 42 was carried out except that the flask wasplaced in a chamber (the capacity of 3000 ml) having a gas feed openingand a gas discharge opening, then the chamber was closed hermetically,and air was so mixed with nitrogen that the concentration of the oxygenin a gas to be supplied to the cells to be cultured became 10% and thegas was supplied through the feed opening at the rate of 25 ml perminute. The results are shown in Table 13.

EXAMPLE 49

The procedure of Example 48 was carried out except that methyl ester ofjasmonic acid was added to give the final concentration of 10⁻⁴ M on the14th day of the culture. The results are shown in Table 13.

COMPARATIVE EXAMPLE 16

The procedure of Example 42 was carried out except that spermidine wasnot added. The results are shown in Tables 13-15.

EXAMPLE 50

The procedure of Example 42 was carried out except that spermin wasadded instead of spermidine to give the final concentration of 10⁻⁹M-1M. The results are shown in Table 14.

EXAMPLE 51

The procedure of Example 42 was carried out except that putrescine wasadded instead of spermin to give the final concentration of 10⁻⁹ M-1M.The results are shown in Table 15.

                                      TABLE 13                                    __________________________________________________________________________           concentration                                                                         concentration                                                                         max oxygen          yield.sup.a) of                      of of methyl concentration growth yield.sup.a) of cephalo- yield.sup.a)                                                            total yield.sup.b)       spermidine jasmonate in the gas rate baccatin III mannine of taxol of                                                              taxane                   (M) (M) phase (%) (times) (mg/l) (mg/l) (mg/l) (mg/l)                       __________________________________________________________________________    Comparative                                                                          0       0       20      2.7  0.14   0.56   0.28 0.98                     Example 16                                                                    Example 42 10.sup.-9 0 20 2.9 0.31 0.58 0.32 1.21                             " 10.sup.-8 0 20 3.1 0.66 1.74 1.99 4.39                                      " 10.sup.-7 0 20 3.1 1.34 5.79 11.76 18.89                                    " 10.sup.-6 0 20 3.2 3.61 6.04 13.51 23.16                                    " 10.sup.-5 0 20 3.1 6.59 7.30 19.28 33.17                                    " 10.sup.-4 0 20 3.1 2.78 6.49 12.96 22.23                                    " 10.sup.-3 0 20 2.9 2.63 4.59 10.09 17.31                                    " 10.sup.-2 0 20 2.0 0.82 2.42 3.89 7.13                                      " 10.sup.-1 0 20 2.1 0.41 1.00 1.94 3.35                                      " 1 0 20 1.4 0.10 0.03 0.08 0.21                                              Example 43 10.sup.-5 0 20 2.9 6.08 6.13 15.24 27.45                           Example 44 10.sup.-5 0 20 2.9 5.46 5.32 10.20 20.98                           Example 45 10.sup.-5 0 20 3.0 2.95 2.90 5.63 11.48                            Example 46 10.sup.-5 0 20 2.9 6.30 6.01 15.43 27.74                           Example 47 10.sup.-5 10.sup.-4 20 2.6 18.96 9.74 21.55 50.25                  Example 48 10.sup.-5 0 10 2.7 13.43 7.81 19.05 40.29                          Example 49 10.sup.-5 10.sup.-4 10 2.6 19.65 8.57 29.67 57.89                __________________________________________________________________________     [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium).                                     [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

                  TABLE 14                                                        ______________________________________                                        concen-             yield.sup.a)                                                                          yield.sup.a) total                                  tration  of of yield.sup.a) yield.sup.b)                                      of growth baccatin cephalo- of of                                             spermin rate III mannine taxol taxane                                         (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)                                     ______________________________________                                        Comparative                                                                           0       2.7     0.14  0.56   0.28  0.98                                 Example 16                                                                    Example 50 10.sup.-9 2.9 0.50 0.64 0.72 1.86                                  " 10.sup.-8 2.3 0.62 1.01 1.79 3.42                                           " 10.sup.-7 2.4 0.68 1.32 2.17 4.17                                           " 10.sup.-6 2.4 0.89 1.97 4.03 6.89                                           " 10.sup.-5 2.1 2.09 5.63 9.46 17.18                                          " 10.sup.-4 2.3 1.64 4.09 6.23 11.96                                          " 10.sup.-3 2.0 0.76 2.68 4.78 8.22                                           " 10.sup.-2 2.1 0.70 1.54 1.06 3.30                                           " 10.sup.-1 1.4 0.39 0.32 0.58 1.29                                           " 1 1.0 0.10 0.04 0.08 0.22                                                 ______________________________________                                         [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium).                                     [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

                  TABLE 15                                                        ______________________________________                                        concen-                                                                         tration  yield.sup.a) yield.sup.a)  total                                     of  of of yield.sup.a) yield.sup.b)                                           putres- growth baccatin cephalo- of of                                        cine rate III mannine taxol taxane                                            (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)                                     ______________________________________                                        Comparative                                                                           0       2.7     0.14  0.56   0.28  0.98                                 Example 16                                                                    Example 51 10.sup.-9 2.8 0.36 0.70 0.69 1.75                                  " 10.sup.-8 2.8 0.40 1.02 1.52 2.94                                           " 10.sup.-7 2.7 1.10 2.31 3.47 6.88                                           " 10.sup.-6 3.0 5.74 5.51 8.19 19.44                                          " 10.sup.-5 3.1 6.98 5.37 11.44 23.79                                         " 10.sup.-4 2.6 6.60 5.25 10.80 22.65                                         " 10.sup.-3 2.2 1.95 1.88 3.07 6.90                                           " 10.sup.-2 1.8 1.13 0.99 1.06 3.18                                           " 10.sup.-1 1.1 0.80 0.97 0.97 2.74                                           " 1 1.0 0.01 0.22 0.13 0.36                                                 ______________________________________                                         [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium).                                     [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

EXAMPLE 52

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent had been added to give the same concentration and shakeculture was carried out with a rotary shaker (amplification of 25 mm,100 rpm) and the callus was subcultured in every 21 days to acceleratethe growth rate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component had been added to give thesame concentration, and shake culture was carried out at 25° C. for 14days. Acetylsalicylic acid (HOOCC₆ H₄ OCOCH₃) was added as antiethyleneagent on the 14th day after starting the culture to give the finalconcentration of 10⁻⁹ M-1M and the culture was further continued foranother 7 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the growth rate thereof. Taxane-type diterpenes wereextracted from the dried cells with methanol and the like, and they weredetermined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpenes. The results are shown in Table 16.

EXAMPLE 53

The procedure of Example 52 was carried out except that acetylsalicylicacid was added at the start (0 day) of the culture to give the finalconcentration of 10⁻⁵ M and the culture was carried out for another 21days. After completing the culture, the procedure of Example 52 wascarried out. The results are shown in Table 16.

EXAMPLE 54

The procedure of Example 52 was carried out except that acetylsalicylicacid was added on the 7th day after starting the culture to give thefinal concentration of 10⁻⁵ M and the culture was further continued foranother 14 days. After completing the culture, the procedure of theExample 52 was carried out. The results are shown in Table 16.

EXAMPLE 55

The procedure of Example 52 was carried out except that acetylsalicylicacid was added on the 18th day after starting the culture to give thefinal concentration of 10⁻⁵ M and the culture was further continued foranother 3 days. After completing the culture, the procedure of theExample 52 was carried out. The results are shown in Table 16.

EXAMPLE 56

The procedure of Example 52 was carried out except that acetylsalicylicacid was added successively a total of 5 times at 2 days' intervalsstarting from the 7th day after starting the culture (to give the finalconcentration of 2×10⁻⁶ M each time, and to give the total concentrationof 10⁻⁵ M). The results are shown in Table 16.

EXAMPLE 57

The procedure of Example 52 was carried out except that methyl ester ofjasmonic acid (which is a compound represented by the general formula(III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³,R₄, R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 andC³ and C⁴ have a double bond between them) was added on the 14th dayafter starting the culture to give the final concentration of 10⁻⁴ M.The results are shown in Table 16.

EXAMPLE 58

The procedure of Example 52 was carried out, except that the flask wasplaced in a chamber (the capacity of 3000 ml) having a gas feed openingand a gas discharge opening, then the chamber was closed hermetically,and air was so mixed with nitrogen that the concentration of the oxygenin a gas to be supplied to the cells to be cultured became 10%, and thegas was supplied through the feed opening at the rate of 25 ml perminute. The results are shown in Table 16.

EXAMPLE 59

The procedure of Example 58 was carried out except that methyl ester ofjasmonic acid was added on the 14th day of the culture to give the finalconcentration of 10⁻⁴ M. The results are shown in Table 16.

COMPARATIVE EXAMPLE 17

The procedure of Example 52 was carried out except that acetylsalicylicacid was not added. The results are shown in Tables 16.

REFERENCE EXAMPLE 1

The procedure of Example 52 was carried out except that Ethrel (C₂ H₆ O₃ClP) of 10⁻³ M was added instead of acetylsalicylic acid, as an ethylenegenerating agent at the start (0 day) of the culture. The results areshown in Table 16.

REFERENCE EXAMPLE 2

The procedure of Example 52 was carried out except that Ethrel of 10⁻³ Mwas added instead of acetylsalicylic acid at the 14th day after startingthe culture. The results are shown in Table 16.

EXAMPLE 60

The procedure of Example 52 was carried out except that aminoxyaceticacid hydrochloride [(H₂ NOCH₂ COOH)₂ HCl] was added as an antiethyleneagent to give the final concentration of 10⁻⁹ M-1M. The results areshown in Table 17.

EXAMPLE 61

The procedure of Example 52 was carried out except that propyl gallate[(HO)₃ C₆ H₂ COOCH₂ CH₂ CH₃ ] was added as an antiethylene agent to givethe final concentration of 10⁻⁹ M-1M. The results are shown in Table 18.

                                      TABLE 16                                    __________________________________________________________________________           concentration                                                                         concentration                                                                        max oxygen                                                                            ethylene       yield.sup.a) of                    of of methyl concentration concen- growth yield.sup.a) of cephalo-                                                                  yield.sup.a)                                                                  total yield.sup.b)      acetylsalicylic jasmonate in the gas tration rate baccatin III mannine                                                              of taxol of                                                                   taxane                  acid (M) (M) phase (%) (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)              __________________________________________________________________________    Comparative                                                                          0       0      20      0   2.7  0.14  0.56  0.28 0.98                    Example 17                                                                    Example 52 10.sup.-9 0 20 0 2.7 0.25 0.62 0.48 1.35                           " 10.sup.-8 0 20 0 2.8 0.79 0.64 0.89 2.32                                    " 10.sup.-7 0 20 0 2.9 0.79 1.27 0.90 2.96                                    " 10.sup.-6 0 20 0 2.5 0.83 2.73 3.00 6.56                                    " 10.sup.-5 0 20 0 2.7 1.52 6.96 13.42 21.90                                  " 10.sup.-4 0 20 0 2.7 1.48 6.54 11.15 19.17                                  " 10.sup.-3 0 20 0 2.6 1.06 3.30 4.44 8.80                                    " 10.sup.-2 0 20 0 2.6 1.01 0.70 1.61 3.32                                    " 10.sup.-1 0 20 0 2.0 0.58 0.12 0.70 1.40                                    " 1 0 20 0 1.5 0.01 0 0.01 0.02                                               Example 53 10.sup.-5 0 20 0 2.6 0.42 1.76 2.98 5.16                           Example 54 10.sup.-5 0 20 0 2.7 0.91 3.55 4.22 8.68                           Example 55 10.sup.-5 0 20 0 2.7 1.22 6.39 9.58 17.19                          Example 56 10.sup.-5 0 20 0 2.6 1.09 5.46 10.02 16.57                         Example 57 10.sup.-5 10.sup.-4 20 0 2.4 11.52 6.60 15.11 33.23                Example 58 10.sup.-5 0 10 0 2.4 7.28 6.46 11.80 25.54                         Example 59 10.sup.-5 10.sup.-4 10 0 2.3 14.79 6.69 18.91 40.39                Reference 0 0 20 10.sup.-3 2.7 0.12 0.14 0.14 0.40                            Example 1                                                                     Reference 0 0 20 10.sup.-3 2.7 0.05 0.16 0.09 0.30                            Example 2                                                                   __________________________________________________________________________     [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

                  TABLE 17                                                        ______________________________________                                        concen-                                                                         tration                                                                       of                                                                            amino-  yield.sup.a) yield.sup.a)  total                                      xy-  of of yield.sup.a) yield.sup.b)                                          acetic growth baccatin cephalo- of of                                         acid rate III mannine taxol taxane                                            (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)                                     ______________________________________                                        Comparative                                                                           0       2.7     0.14  0.56   0.28  0.98                                 Example 17                                                                    Example 60 10.sup.-9 2.3 0.21 0.58 0.59 1.38                                  " 10.sup.-8 1.8 0.61 0.62 0.77 2.00                                           " 10.sup.-7 1.7 0.66 0.65 1.02 2.33                                           " 10.sup.-6 1.5 1.08 0.73 1.19 3.00                                           " 10.sup.-5 1.7 1.30 1.16 1.35 3.81                                           " 10.sup.-4 1.7 1.92 1.22 2.66 5.80                                           " 10.sup.-3 1.5 1.11 0.71 1.61 3.43                                           " 10.sup.-2 1.5 0.32 0.71 0.90 1.93                                           " 10.sup.-1 1.2 0.33 0.26 0.44 1.03                                           " 1 0.9 0 0 0 0                                                             ______________________________________                                         [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium).                                     [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

                  TABLE 18                                                        ______________________________________                                        concen-                                                                         tration  yield.sup.a) yield.sup.a)  total                                     of  of of yield.sup.a) yield.sup.b)                                           propyl growth baccatin cephalo- of of                                         gallate rate III mannine taxol taxane                                         (M) (times) (mg/l) (mg/l) (mg/l) (mg/l)                                     ______________________________________                                        Comparative                                                                           0       2.7     0.14  0.56   0.28  0.98                                 Example 17                                                                    Example 61 10.sup.-9 2.7 0.41 0.59 0.37 1.37                                  " 10.sup.-8 2.7 0.42 0.68 0.74 1.74                                           " 10.sup.-7 2.7 0.55 0.72 1.52 2.79                                           " 10.sup.-6 3.1 1.91 4.38 7.66 13.95                                          " 10.sup.-5 3.0 2.82 5.07 8.24 16.13                                          " 10.sup.-4 3.0 0.87 4.42 6.98 12.27                                          " 10.sup.-3 3.2 0.84 4.30 6.96 12.10                                          " 10.sup.-2 2.9 0.69 2.01 4.33 7.03                                           " 10.sup.-1 1.9 0.48 0.25 0.65 1.38                                           " 1 1.1 0.01 0 0 0.01                                                       ______________________________________                                         [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium).                                     [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

EXAMPLE 62

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent had been added to give the same concentration and shakeculture was carried out with a rotary shaker (amplification of 25 mm,100 rpm) and the callus was subcultured in every 21 days to acceleratethe growth rate of the callus.

One gram (fresh weight) of the cultured cells thus obtained wasinoculated to an Erlenmeyer flask containing 20 ml of liquid Woody PlantMedium to which the above-mentioned component had been added to give thesame concentration, and the flask was placed in a chamber (the capacityof 3000 ml) having a gas feed opening and a gas discharge opening, thenthe chamber was closed hermetically, and air was so mixed with nitrogenthat the concentration of the oxygen in a gas to be supplied to thecells to be cultured became 4-15%, and while the gas was suppliedthrough the feed opening at the rate of 25 ml per minute, shake culturewas carried out at 25° C. for 21 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the growth rate thereof. Taxane-type diterpenes wereextracted from the dried cells with methanol and the like, and they weredetermined by comparing with standard taxol, cephalomannine, andbaccatin III using high performance liquid chromatography to measure theyields of the taxane-type diterpenes. The results are shown in Table 19.

COMPARATIVE EXAMPLE 18

The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 20%. The results are shown in Table 19.

REFERENCE EXAMPLE 3

The procedure of Example 62 was carried out except that the culturedcells inoculated to the flask were cultured in the atmosphere. Theresults are shown in Table 19.

EXAMPLE 63

The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 10% and the mixed gas was supplied fromthe start of the culture for 3 days, then air was supplied till the endof the culture (for 18 days). The results are shown in Table 19.

EXAMPLE 64

The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 10% and the mixed gas was supplied fromthe start of the culture for 7 days, then air was supplied till the endof the culture (for 14 days). The results are shown in Table 19.

EXAMPLE 65

The procedure of Example 62 was carried out except that theconcentration of the oxygen in a gas to be supplied to the cells to becultured was controlled to be 10%, and the mixed gas was supplied fromthe start of the culture for 14 days, then air was supplied till the endof the culture (for 7 days). The results are shown in Table 19.

EXAMPLE 66

The procedure of Example 62 was carried out except that methyl ester ofjasmonic acid (which is a compound represented by the general formula(III) wherein R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R², R³,R⁴, R⁵, and R⁶ are hydrogen atoms, R⁷ is methoxy group, n is 1 and C³and C⁴ have a double bond between them; 90% of which is in thetrans-form, and 10% of which is in the cis-form) was added as one ofjasmonic acids on the 14th day after starting the culture to give thefinal concentration of 10-1000 μM. The results are shown in Table 20.The productivity of taxane-type diterpene could be remarkably improvedby combination of supply of oxygen of low concentration and addition ofthe methyl ester of jasmonic acid.

EXAMPLE 67

Eighty-five gram (fresh weight) of cultured cells obtained in Example 62with accelerated growth rate were inoculated in a tank for stirred tankculture (capacity of 3000 ml) having an electrode for a dissolved oxygenconcentration and a dissolved oxygen concentration controller, intowhich 1700 ml of liquid Woody Plant Medium had been poured. Then stirredtank culture was carried out at 25° C. for 21 days while the dissolvedoxygen concentration in the medium was controlled to be 0.1 ppm or lessby adjusting the mixing ratio of air and nitrogen. The schematic diagramof the culture apparatus is shown in FIG. 3 and the results are shown inTable 21.

EXAMPLE 68

The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 1 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

EXAMPLE 69

The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 2 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

EXAMPLE 70

The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 4 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

EXAMPLE 71

The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen was controlled to be 6 ppm or lessby adjusting the mixing ratio. The results are shown in Table 21.

COMPARATIVE EXAMPLE 19

The procedure of Example 67 was carried out except that air wassupplied. The results are shown in Table 21.

EXAMPLE 72

The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen in the medium was controlled to be4 ppm or less from the start of the culture for 3 days by adjusting themixing ratio, and air was supplied till the end of the culture (for 18days). The results are shown in Table 21.

EXAMPLE 73

The procedure of Example 67 was carried out except that theconcentration of the dissolved oxygen in the medium was controlled to be4 ppm or less from the start of the culture for 7 days by adjusting themixing ratio, and air was supplied till the end of the culture (for 14days). The results are shown in Table 21.

EXAMPLE 74

The procedure of Example 67 was carried out, except that theconcentration of the dissolved oxygen in the medium was controlled to be4 ppm or less from the start of the culture for 14 days by adjusting themixing ratio, and air was supplied till the end of the culture (for 7days). The results are shown in Table 21.

                  TABLE 19                                                        ______________________________________                                        max                                                                             oxygen                                                                        concen-                                                                       tration  yield.sup.a) yield.sup.a)  total                                     in  of of yield.sup.a) yield.sup.b)                                           the gas growth baccatin cephalo- of of                                        phase rate III mannine taxol taxane                                           (%) (times) (mg/l) (mg/l) (mg/l) (mg/l)                                     ______________________________________                                        Comparative                                                                           20      3.01    0.78  1.71   1.46  3.95                                 Example 18                                                                    Example 62  4 2.09 1.65 5.24 1.78 8.67                                        "  7 2.60 3.37 7.25 2.49 13.11                                                " 10 2.90 3.19 7.51 2.96 13.66                                                " 15 2.82 3.18 6.86 2.18 12.22                                                Example 63 10.sup.c) 2.79 1.33 4.88 1.74 7.95                                 Example 64 10.sup.c) 2.62 3.21 7.16 1.75 12.12                                Example 65 10.sup.c) 2.66 3.17 7.16 1.92 12.25                                Reference 20 3.03 0.77 1.23 0.95 2.95                                         Example 3                                                                   ______________________________________                                         [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                      [.sup.c) : The value shows the max oxygen concentration in the gas phase      in the chamber during the mixed gas supply period.                       

                  TABLE 19                                                        ______________________________________                                        max                                                                             oxygen                                                                        concen-                                                                       tration  yield.sup.a) yield.sup.a)  total                                     in  of of yield.sup.a) yield.sup.b)                                           the gas growth baccatin cephalo- of of                                        phase rate III mannine taxol taxane                                           (%) (times) (mg/l) (mg/l) (mg/l) (mg/l)                                     ______________________________________                                        Comparative                                                                           20      3.01    0.78  1.71   1.46  3.95                                 Example 18                                                                    Example 62  4 2.09 1.65 5.24 1.78 8.67                                        "  7 2.60 3.37 7.25 2.49 13.11                                                " 10 2.90 3.19 7.51 2.96 13.66                                                " 15 2.82 3.18 6.86 2.18 12.22                                                Example 63 10.sup.c) 2.79 1.33 4.88 1.74 7.95                                 Example 64 10.sup.c) 2.62 3.21 7.16 1.75 12.12                                Example 65 10.sup.c) 2.66 3.17 7.16 1.92 12.25                                Reference 20 3.03 0.77 1.23 0.95 2.95                                         Example 3                                                                   ______________________________________                                         [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                      [.sup.c) : The value shows the max oxygen concentration in the gas phase      in the chamber during the mixed gas supply period.                       

                                      TABLE 20                                    __________________________________________________________________________           max oxygen                                                                            concentration            yield.sup.a) of                         concentration of methyl growth yield.sup.a) of yield.sup.a) cephalo-                                                      total yield.sup.b)                in gas phase jasmonate rate baccatin III of taxol mannine of taxane                                                        (%) (μM) (times) (mg/l)                                                   (mg/l) (mg/l) (mg/l)            __________________________________________________________________________    Comparative                                                                          20      0       3.01 0.78   1.71 1.46  3.95                              Example 18                                                                    Example 62 10 0 2.90 3.19 7.51 2.96 13.66                                     Example 66 10 10 2.82 2.26 10.24 1.73 14.73                                   " 10 100 2.45 11.32 22.44 2.29 36.05                                          " 10 1000 1.47 2.10 5.04 1.47 8.61                                          __________________________________________________________________________     [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                 

                                      TABLE 21                                    __________________________________________________________________________           max dissolved                                                            oxygen    yield.sup.a) of                                                     concentra- growth yield.sup.a) of yield.sup.a) cephalo- total yield.sup.                                         b)                                         tion in the rate baccatin III of taxol mannine of taxane                      medium (ppm) (times) (mg/l) (mg/l) (mg/l) (mg/l)                            __________________________________________________________________________    Comparative                                                                          8  (100.sup.c))                                                                      3.01 0.58   1.74 0.91  3.23                                       Example 19                                                                    Example 67 0.1 (1.25.sup.c)) 2.20 0.70 2.05 1.12 3.87                         Example 68 1   (12.5.sup.c)) 2.25 1.05 3.69 1.22 5.96                         Example 69 2   (25.sup.c)) 2.49 2.44 5.54 2.70 10.68                          Example 70 4   (50.sup.c)) 2.75 3.91 8.14 1.80 13.85                          Example 71 6   (75.sup.c)) 2.98 2.52 5.69 1.23 9.44                           Example 72 4.sup.d) (50.sup.e)) 2.89 1.62 4.02 1.54 7.18                      Example 73 4.sup.d) (50.sup.e)) 2.62 3.32 8.46 3.08 14.86                     Example 74 4.sup.d) (50.sup.e)) 2.75 3.19 7.72 2.65 13.56                   __________________________________________________________________________     [.sup.a) : The yield was calculated based on the total amount of              production (in the cell + in the medium)                                      [.sup.b) : The total yield was calculated by adding the yield of baccatin     III, the yield of cephalomannine and the yield of taxol.                      [.sup.c) : The figure in the parentheses shows the ratio (in %) of the ma     dissolved oxygen concentration in the comparative example and in each         example with respect to the saturated dissolved oxygen concentration (8       ppm) at 25° C.                                                         [.sup.d) : The value shows the max dissolved oxygen concentration in the      culture medium during the mixed gas supply period.                            [.sup.e) : The figure in the parentheses shows the ratio (in %) of the ma     dissolved oxygen concentration during the mixed gas supply period in each     example with respect to the saturated dissolved oxygen concentration (8       ppm) at 25° C.                                                    

EXAMPLE 75

A part of stem of Taxus baccata LINN which had been previouslysterilized with 2% antiformin solution or 70% ethanol solution and thelike, was placed on solid Woody Plant Medium (containing gelan gum of0.25% by weight) to which naphthalenacetic acid had been added to givethe concentration of 10⁻⁵ M, and static culture was carried out at 25°C. in a dark place to provide Taxus baccata LINN callus. One gram (freshweight) of the callus was inoculated to an Erlenmeyer flask containing20 ml of liquid Woody Plant Medium to which the above-mentionedcomponent had been added to give the same concentration and shakeculture was carried out with a rotary shaker (amplification of 25 mm,100 rpm), and the callus was subcultured in every 21 days to acceleratethe growth rate thereof.

One gram (fresh weight) of the cultured cells thus obtained wasfractionated firstly by a stainless steel mesh and cell clusters havingthe size of 250-840 μm were obtained. A density gradient with specificgravities of 1.03, 1.05, 1.07, 1.09 and 1.11 (g/ml) was produced by theuse of Ficoll and the above-mentioned cells were layered over it andcentrifuged at 700 rpm for 6 minutes. The cells were fractionated intoeach layer according to the difference of the specific gravity. Thecells contained in each layer were so fractionated that they were notmixed each other and washed with 2% sucrose solution three times ormore, to wash off Ficoll. After the washing, about 0.1 g (fresh weight)of the cells were transferred to a culture well having an inner diameterof 18 mm, containing 0.8 ml of liquid Woody Plant Medium and shakeculture was carried out at 25° C. for 21 days. After culturing them for21 days, the whole amount of the cells was transferred to a culture wellhaving an inner diameter of 36 mm containing 3 ml of the above-mentionedliquid medium and shake culture was further continued at 25° C. foranother 28 days.

After completing the culture, cultured cells of Taxus baccata LINN wereharvested by filtration and lyophilized, then the dry weight wasmeasured to obtain the growth weight thereof per litre the liquidmedium. Taxane-type diterpenes were extracted from the dried callus withmethanol and the like, and they were determined by comparing withstandard taxol, cephalomannine, and baccatin III using high performanceliquid chromatography to measure the yields of the taxane-typediterpenes. The results are shown in Table 22, FIG. 4 and FIG. 5.

COMPARATIVE EXAMPLE 20

The procedure of Example 75 was carried out except that fractionationaccording to the density gradient was not carried out after separationof the cell clusters by the stainless mesh. The results are shown inTable 22, FIG. 4 and FIG. 5.

EXAMPLE 76

The procedure of Example 75 was carried out except that the culturedcells which have the same parent plant, but were induced to callus indifferent stage were used. Provided that the cells contained in a layerhaving the specific gravity of 1.07 or more were collected in one groupand cultured. The results are shown in Table 22.

COMPARATIVE EXAMPLE 21

The procedure of Example 76 was carried out except that fractionationaccording to the density gradient was not carried out after separationof the cell clusters by the stainless mesh. The results are shown inTable 22.

                  TABLE 22                                                        ______________________________________                                                                             content*) of                               specific cell content*) of content*) cephalo-                                 gravity yield baccatin III of taxol mannine                                   range (g/l) (ppm) (ppm) (ppm)                                               ______________________________________                                        Comparative                                                                           --       12.6   1.5     13.0   27.5                                     Example 20                                                                    Example 75 <1.03 12.5 7.9 184.0 170.3                                          1.03-1.05 12.5 32.1 90.8 123.7                                                1.05-1.07 12.2 16.3 93.5 114.1                                                1.07-1.09 12.6 0.0 3.9 12.3                                                   1.09-1.11 11.9 0.0 2.1 8.6                                                    1.11< 13.5 0.0 2.5 6.3                                                       Comparative -- 9.3 256.5 864.0 662.5                                          Example 21                                                                    Example 76 1.03-1.05 9.0 151.3 1660.5 1050.2                                   1.05-1.07 9.9 155.7 1140.8 804.0                                              1.07< 9.5 19.0 333.1 161.3                                                 ______________________________________                                         *): [The content was calculated by dividing the total amount of productio     (in the cell + in the medium) by the cell yield                          

REFERENCE EXAMPLE 4

About 0.2 g (fresh weight) of the cells obtained in Example 75 whichwere cultured after being fractionated into a layer having the specificgravity range of 1.03 or less (Table 22) were inoculated to a culturewell having an inner diameter of 36 mm containing 3 ml of liquid WoodyPlant Medium and shake culture was carried out at 25° C. for another 28days. After completing the culture, the cells were again fractionated bythe density gradient with specific gravities of 1.03, 1.05, 1.07, 1.09and 1.11 (g/ml). Immediately after the density gradient fractionation,the cells were collected and the distribution of the fractionated cellsand the contents of the taxane-type diterpenes were determined. Theresults are shown in Table 23, FIG. 6 and FIG. 7.

                  TABLE 23                                                        ______________________________________                                                    distri-  content*)       content*) of                               specific bution of content*) cephalo-                                         gravity of cells baccatin of taxol mannine                                    range (%) III (ppm) (ppm) (ppm)                                             ______________________________________                                        Reference                                                                            <1.03    20.2     13.9   123.9  66.5                                     Example 4                                                                      1.03-1.05 29.8 28.1 216.1 128.7                                               1.05-1.07 15.1 71.7 315.5 226.0                                               1.07-1.09 8.1 96.2 475.8 382.6                                                1.09-1.11 6.2 123.5 619.4 511.8                                               1.11< 20.5 195.2 609.6 460.2                                               ______________________________________                                         *): [The content was calculated by dividing the amount of production in       the cell by the cell yield.                                              

EXAMPLE 77

One gram (fresh weight) of the same cultured cells that were used inExample 1 was inoculated to an Erlenmeyer flask containing 20 ml of aliquid containing 10⁻⁵ M-10⁻² M of potassium peroxodisulfate and shakenat 25° C. for 21 days to carry out the first stage of the culture.

After completing the culture, the cultured cells were harvested byfiltration and a part of the cells were used as seed cells for thesecond stage of the culture and the rest of the cells were subjected tothe measurement of the cell yield and the taxane content in the cells.Accordingly, 1 g (fresh weight) of the cultured cells were inoculated toan Erlenmeyer flask containing 20 ml of liquid Woody Plant Medium towhich naphthalenacetic acid had been added to give the concentration of10⁻⁵ M and shake culture was carried out at 25° C. for 14 days. On the14th day of the culture, methyl jasmonate was added to the medium togive the concentration in the medium of 100 μM and the culture wasfurther continued for another 7 days. On the other hand, the rest of thecells obtained in the first stage of the culture were lyophilized, thenthe dry weight was measured to obtain the cell yield thereof per litreof the liquid medium. The content of taxol in the dried cells wasmeasured by high performance liquid chromatography. The yield of thecells and the yield of taxol were measured for the cells obtained bysecond stage of the culture in the same manner as that for the cellsobtained in the first stage of the culture. The results are shown inTable 24.

COMPARATIVE EXAMPLE 22

The procedure of Example 77 was carried out except that potassiumperoxodisulfate was not used. The results are shown in Table 24.

                                      TABLE 24                                    __________________________________________________________________________           additives to be used in                                                  the first stage of the                                                        cultivation result of the first result of the second                               concentration of                                                                            stage of the culture                                                                     stage of the culture                                 potassium     yield of                                                                           content of                                                                          yield of                                                                           yield of                                   peroxodisulfate time for cells taxol cells taxol                              in the medium addition (g/l) (ppm) (g/l) (mg/l)                             __________________________________________________________________________    Example 77                                                                           10.sup.-5 M                                                                            1st day                                                                            17.1 97    15.6 14.0                                       " 10.sup.-4 M " 16.2 250 15.0 20.4                                            " 5 × 10.sup.-4 M " 15.5 183 14.4 16.3                                  " 10.sup.-3 M " 15.2 84 13.0 13.6                                             " 5 × 10.sup.-3 M " 14.0 45 12.1 11.7                                   Comparative -- -- 16.5 53 15.4 10.1                                           Example 22                                                                  __________________________________________________________________________

EXAMPLE 78

A hundred grams (fresh weight) of the same cultured cells that were usedin Example 1 were inoculated in a tank f or stirred tank culture(capacity of 2 liter; FIG. 8) f illed with 1 liter of standard liquidWoody Plant Medium (sucrose concentration: 20 g/l, nitrate ionconcentration: 14.7 mM, α-naphthalenacetic acid: 10⁻⁵ M) to which 2 mm[Ag(S₂ O₃)₂ ]³⁻ had been added, and the culture was started at 25° C. inthe dark at the agitation rate of 40 rpm, while air was fed at 0.1 literper minute, and a medium containing 20 g/l of sucrose and 20 mM ofsodium nitrate was supplied continuously in a period starting from thesecond day till the 14th day of the culture in such a manner that theamount of the sucrose added in one day became 2 g/l and the amount ofnitrate ion added in one day became 2 mmol/l, and the culture solutionwas continuously taken out through a discharge opening, which wasdifferent from the nutrition source feed opening, and to which astainless filter of 100 mesh was attached, at the same rate as that ofthe addition of the nutrition source solution (the medium renewing ratioin the culture vessel was 10% per day) to carry out stirred tank culturefor 21 days. After completing the culture, the cultured cells and themedium were collected and the yield of taxol was measured in the samemanner as that used in the said Example 1. The results are shown inTable 25.

COMPARATIVE EXAMPLE 23

The procedure of Example 78 was carried out except that the nutritionsource was not added midway. The results are shown in Table 25.

                  TABLE 25                                                        ______________________________________                                        yield of cells  yield of taxol (mg/l)                                         (g dried weight /l)                                                                           from cells                                                                              from medium                                                                              total                                    ______________________________________                                        Example 78                                                                            38          31        89       125                                      Comparative 22 33 27  60                                                      Example 23                                                                  ______________________________________                                    

EXAMPLE 79

Fifty grams (fresh weight) of the same cultured cells that were used inExample 1, and 1 liter of liquid Woody Plant Medium were transferred toa culture tank (capacity of 2 liter) and the culture was carried out at25° C. in the dark at the agitation rate of 40 rpm, at the aerationspeed of 0.1 liter per minute for 14 days. The precipitated cell volume(PCV) measured on the 14th day after starting the culture was 0.2 liter.From the 14th day, supply of a fresh medium, wherein 3mM [Ag(S₂ O₃)₂ ]³⁻had been added to the medium of the same composition as that of theinitial medium, as well as taking out of a culture solution which wasfree from cells were started. The amount of the fresh medium to besupplied per day was 2/5 of the PCV at that time and the amount of theculture solution free from the cells to be taken out was controlled tobe 1 liter. On the 35th day after starting the culture, PCV reached 0.6liter. After that, the stationary state was maintained by taking out theculture solution containing cells once a day to keep the average PCV to0.6 liter and by taking out the culture solution free from cells to keepthe amount of the culture solution to 1 liter. The culture was carriedout for 90 days after the start of the culture. The amount of the freshmedium supplied during the stationary state of 60 days was 15 liters,the amount of the medium taken out of the culture tank was 14 liters andthe amount of the cells obtained was 0.15 kg (dry weight) and thespecific growth rate μ was 0.08 (day⁻¹), the average medium renewingratio was 2.88. The results of the analysis of cells and medium takenout from the culture tank under stationary state showed that 525 mg oftaxol was produced. That was equal to the productivity of 8.8mg/liter/day.

The amount of the taxol contained in the cells and the medium weremeasured in the same manner as that used in Example 1.

EXAMPLE 80

Fifty grams (fresh weight) of the same cultured cells that were used inExample 1 and 1 liter of liquid Woody Plant Medium were transferred to aculture tank (capacity of 2 liters) and the culture was carried out inthe dark, at the agitation rate of 40 rpm, at 25° C. for 14 days, whilean air to which 2% carbon dioxide gas had been added was fed at 0.1liter per minute. After completing the culture, the cells and the mediumwere collected and 15.2 g of dry cells were obtained. Determination ofthe amount of taxol contained in the cells and in the medium which wascarried out in the same manner as that used in Example 1 showed that 31mg of taxol was produced.

COMPARATIVE EXAMPLE 24

The procedure of Example 1 was carried out except that jasmone was addedinstead of methyl tuberonate to give the final concentration of 0.1-1000μM. The results are shown in Table 26.

COMPARATIVE EXAMPLE 25

The procedure of Comparative Example 24 was carried out except thatjasmone was not added. The results are shown in Table 26.

                  TABLE 26                                                        ______________________________________                                                               yield*)        yield*)                                   concentration  of yield*) of                                                  of cell baccatin of cephalo-                                                  jasmone yield III taxol mannine                                               (μM) (g/l) (mg/l) (mg/l) (mg/l)                                          ______________________________________                                        Comparative                                                                           0.1        12.2    0.3    3.0   1.2                                     Example 24                                                                    Comparative 1 12.1 0.4 3.2 1.0                                                Example 24                                                                    Comparative 10 11.3 0.3 3.3 0.8                                               Example 24                                                                    Comparative 100 11.3 0.3 3.2 0.6                                              Example 24                                                                    Comparative 1000 10.9 0.2 2.5 0.8                                             Example 24                                                                    Comparative 0 12.2 0.2 2.8 1.5                                                Example 25                                                                  ______________________________________                                         [*): The yield was calculated based on the total amount of production (in     the cell + in the medium)                                                

Industrial Applicability

The present invention allows industrial production of a taxane-typediterpene including taxol which is useful as a therapeutic agent forovarian cancer, mammary cancer, lung cancer and the like.

We claim:
 1. A method of producing a taxane ring-containing alkaloidcompound comprising culturing tissues or cells of a plant, belonging tothe genus Taxus and which produce a taxane ring-containing alkaloidcompound, in a nutrient medium and in contact with a gas phasecomprising oxygen and from about 0.1 to 5% of carbon dioxide;andrecovering the taxane ring-containing alkaloid compound from theculture.